Histone deacetylase 8 promotes innate antiviral immunity through deacetylation of RIG-I.

Histone deacetylates family proteins have been studied for their function in regulating viral replication by deacetylating non-histone proteins. RIG-I (Retinoic acid-inducible gene I) is a critical protein in RNA virus-induced innate antiviral signaling pathways. Our previous research showed that HDAC8 (histone deacetylase 8) involved in innate antiviral immune response, but the underlying mechanism during virus infection is still unclear. In this study, we showed that HDAC8 was involved in the regulation of vesicular stomatitis virus (VSV) replication. Over-expression of HDAC8 inhibited while knockdown promoted VSV replication. Further exploration demonstrated that HDAC8 interacted with and deacetylated RIG-I, which eventually lead to enhance innate antiviral immune response. Collectively, our data clearly demonstrated that HDAC8 inhibited VSV replication by promoting RIG-I mediated interferon production and downstream signaling pathway.

Development of Robust Freeze-Drying Process for Long-Term Stability of rVSV-SARS-CoV-2 Vaccine.

The thermostability of vaccines, particularly enveloped viral vectored vaccines, remains a challenge to their delivery wherever needed. The freeze-drying of viral vectored vaccines is a promising approach but remains challenging due to the water removal process from the outer and inner parts of the virus. In the case of enveloped viruses, freeze-drying induces increased stress on the envelope, which often leads to the inactivation of the virus. In this study, we designed a method to freeze-dry a recombinant vesicular stomatitis virus (VSV) expressing the SARS-CoV-2 spike glycoprotein. Since the envelope of VSV is composed of 50% lipids and 50% protein, the formulation study focused on both the protein and lipid portions of the vector. Formulations were prepared primarily using sucrose, trehalose, and sorbitol as cryoprotectants; mannitol as a lyoprotectant; and histidine as a buffer. Initially, the infectivity of rVSV-SARS-CoV-2 and the cake stability were investigated at different final moisture content levels. High recovery of the infectious viral titer (~0.5 to 1 log loss) was found at 3-6% moisture content, with no deterioration in the freeze-dried cakes. To further minimize infectious viral titer loss, the composition and concentration of the excipients were studied. An increase from 5 to 10% in both the cryoprotectants and lyoprotectant, together with the addition of 0.5% gelatin, resulted in the improved recovery of the infectious virus titer and stable cake formation. Moreover, the secondary drying temperature of the freeze-drying process showed a significant impact on the infectivity of rVSV-SARS-CoV-2. The infectivity of the vector declined drastically when the temperature was raised above 20 °C. Throughout a long-term stability study, formulations containing 10% sugar (sucrose/trehalose), 10% mannitol, 0.5% gelatin, and 10 mM histidine showed satisfactory stability for six months at 2-8 °C. The development of this freeze-drying process and the optimized formulation minimize the need for a costly cold chain distribution system.

Development of a vesicular stomatitis virus pseudotyped with herpes B virus glycoproteins and its application in a neutralizing antibody detection assay.

Herpes B virus (BV) is a zoonotic virus and belongs to the genus Simplexvius, the same genus as human herpes simplex virus (HSV). BV typically establishes asymptomatic infection in its natural hosts, macaque monkeys. However, in humans, BV infection causes serious neurological diseases and death. As such, BV research can only be conducted in a high containment level facility (i.e., biosafety level [BSL] 4), and the mechanisms of BV entry have not been fully elucidated. In this study, we generated a pseudotyped vesicular stomatitis virus (VSV) expressing BV glycoproteins using G-complemented VSV∆G system, which we named VSV/BVpv. We found that four BV glycoproteins (i.e., gB, gD, gH, and gL) were required for the production of a high-titer VSV/BVpv. Moreover, VSV/BVpv cell entry was dependent on the binding of gD to its cellular receptor nectin-1. Pretreatment of Vero cells with endosomal acidification inhibitors did not affect the VSV/BVpv infection. The result indicated that VSV/BVpv entry occurred by direct fusion with the plasma membrane of Vero cells and suggested that the entry pathway was similar to that of native HSV. Furthermore, we developed a VSV/BVpv-based chemiluminescence reduction neutralization test (CRNT), which detected the neutralization antibodies against BV in macaque plasma samples with high sensitivity and specificity. Crucially, the VSV/BVpv generated in this study can be used under BSL-2 condition to study the initial entry process through gD-nectin-1 interaction and the direct fusion of BV with the plasma membrane of Vero cells.IMPORTANCEHerpes B virus (BV) is a highly pathogenic zoonotic virus against humans. BV belongs to the genus Simplexvius, the same genus as human herpes simplex virus (HSV). By contrast to HSV, cell entry mechanisms of BV are not fully understood. The research procedures to manipulate infectious BV should be conducted in biosafety level (BSL)-4 facilities. As pseudotyped viruses provide a safe viral entry model because of their inability to produce infectious progeny virus, we tried to generate a pseudotyped vesicular stomatitis virus bearing BV glycoproteins (VSV/BVpv) by modification of expression constructs of BV glycoproteins, and successfully obtained VSV/BVpv with a high titer. This study has provided novel information for constructing VSV/BVpv and its usefulness to study BV infection.

The interferon-induced protein, IFIT2, requires RNA-binding activity and neuronal expression to protect mice from intranasal vesicular stomatitis virus infection.

The interferon (IFN) system protects mammals from diseases caused by virus infections. IFN synthesis is induced by pattern recognition receptor signaling pathways activated by virus infection. IFN is secreted from the infected cells and acts upon neighboring cells by binding cell surface receptors and triggering induction of hundreds of IFN-stimulated genes and proteins, many of which block different steps of virus replication. The IFN-induced tetratricopeptide repeat proteins (IFIT) are a family of RNA-binding proteins. We and others have previously reported that IFIT2 protects mice from many neurotropic RNA viruses; indeed, Ifit2-/- mice are very susceptible to intranasal or subcutaneous infections with vesicular stomatitis virus (VSV). Here, using a newly generated conditional knockout mouse, we report that ablation of Ifit2 expression only in neuronal cells was sufficient to render mice susceptible to neuropathogenesis caused by intranasal, but not subcutaneous, infection of VSV. Another genetically modified mouse line, expressing a mutant IFIT2 that cannot bind RNA, was as susceptible to VSV infection as Ifit2-/- mice. These results demonstrated that IFIT2 RNA-binding activity is essential for protecting mice against neurological diseases caused by intranasal infection of VSV.IMPORTANCEInterferon's (IFN's) antiviral effects are mediated by the proteins encoded by the interferon-stimulated genes. IFN-stimulated genes (IFIT2) is one such protein, which inhibits replication of many RNA viruses in the mouse brain and the resultant neuropathology. Our study sheds light on how IFIT2 works. By ablating Ifit2 expression only in neuronal cells, using a newly generated conditional knockout mouse line, we showed that Ifit2 induction in the neurons of the infected mouse was necessary for antiviral function of interferon. IFIT2 has no known enzyme activity; instead, it functions by binding to cellular or viral proteins or RNAs. We engineered a new mouse line that expressed a mutant IFIT2 that cannot bind RNA. These mice were very susceptible to infection with vesicular stomatitis virus indicating that the RNA-binding property of IFIT2 was essential for its antiviral function in vivo.

Octyl itaconate enhances VSVΔ51 oncolytic virotherapy by multitarget inhibition of antiviral and inflammatory pathways.

The presence of heterogeneity in responses to oncolytic virotherapy poses a barrier to clinical effectiveness, as resistance to this treatment can occur through the inhibition of viral spread within the tumor, potentially leading to treatment failures. Here we show that 4-octyl itaconate (4-OI), a chemical derivative of the Krebs cycle-derived metabolite itaconate, enhances oncolytic virotherapy with VSVΔ51 in various models including human and murine resistant cancer cell lines, three-dimensional (3D) patient-derived colon tumoroids and organotypic brain tumor slices. Furthermore, 4-OI in combination with VSVΔ51 improves therapeutic outcomes in a resistant murine colon tumor model. Mechanistically, we find that 4-OI suppresses antiviral immunity in cancer cells through the modification of cysteine residues in MAVS and IKKß independently of the NRF2/KEAP1 axis. We propose that the combination of a metabolite-derived drug with an oncolytic virus agent can greatly improve anticancer therapeutic outcomes by direct interference with the type I IFN and NF-κB-mediated antiviral responses.

Natural IgG protects against early dissemination of vesicular stomatitis virus.

Neonatal Fc receptor (FcRn) recycles immunoglobulin G, and inhibition of FcRn is used clinically for treatment of autoimmune diseases. In this work, using the vesicular stomatitis virus (VSV) mouse infection model system, we determined the role of FcRn during virus infection. While induction of neutralizing antibodies and long-term protection of these antibodies was hardly affected in FcRn deficient mice, FcRn deficiency limited the amount of natural IgG (VSV-specific) antibodies. Lack of natural antibodies (nAbs) limited early control of VSV in macrophages, accelerated propagation of virus in several organs, led to the spread of VSV to the neural tissue resulting in fatal outcomes. Adoptive transfer of natural IgG into FcRn deficient mice limited early propagation of VSV in FcRn deficient mice and enhanced survival of FcRn knockout mice. In line with this, vaccination of FcRn mice with very low dose of VSV prior to infection similarly prevented death after infection. In conclusion we determined the importance of nAbs during VSV infection. Lack of FcRn limited nAbs and thereby enhanced the susceptibility to virus infection.

Fusogenic vesicular stomatitis virus combined with natural killer T cell immunotherapy controls metastatic breast cancer.

BACKGROUND: Metastatic breast cancer is a leading cause of cancer death in woman. Current treatment options are often associated with adverse side effects and poor outcomes, demonstrating the need for effective new treatments. Immunotherapies can provide durable outcomes in many cancers; however, limited success has been achieved in metastatic triple negative breast cancer. We tested whether combining different immunotherapies can target metastatic triple negative breast cancer in pre-clinical models. METHODS: Using primary and metastatic 4T1 triple negative mammary carcinoma models, we examined the therapeutic effects of oncolytic vesicular stomatitis virus (VSVΔM51) engineered to express reovirus-derived fusion associated small transmembrane proteins p14 (VSV-p14) or p15 (VSV-p15). These viruses were delivered alone or in combination with natural killer T (NKT) cell activation therapy mediated by adoptive transfer of α-galactosylceramide-loaded dendritic cells. RESULTS: Treatment of primary 4T1 tumors with VSV-p14 or VSV-p15 alone increased immunogenic tumor cell death, attenuated tumor growth, and enhanced immune cell infiltration and activation compared to control oncolytic virus (VSV-GFP) treatments and untreated mice. When combined with NKT cell activation therapy, oncolytic VSV-p14 and VSV-p15 reduced metastatic lung burden to undetectable levels in all mice and generated immune memory as evidenced by enhanced in vitro recall responses (tumor killing and cytokine production) and impaired tumor growth upon rechallenge. CONCLUSION: Combining NKT cell immunotherapy with enhanced oncolytic virotherapy increased anti-tumor immune targeting of lung metastasis and presents a promising treatment strategy for metastatic breast cancer.

Establishment of replication-competent vesicular stomatitis virus recapitulating SADS-CoV entry.

Zoonotic coronaviruses pose a continuous threat to human health, with newly identified bat-borne viruses like swine acute diarrhea syndrome coronavirus (SADS-CoV) causing high mortality in piglets. In vitro studies indicate that SADS-CoV can infect cell lines from diverse species, including humans, highlighting its potential risk to human health. However, the lack of tools to study viral entry, along with the absence of vaccines or antiviral therapies, perpetuates this threat. To address this, we engineered an infectious molecular clone of Vesicular Stomatitis Virus (VSV), replacing its native glycoprotein (G) with SADS-CoV spike (S) and inserting a Venus reporter at the 3' leader region to generate a replication-competent rVSV-Venus-SADS S virus. Serial passages of rVSV-Venus-SADS S led to the identification of an 11-amino-acid truncation in the cytoplasmic tail of the S protein, which allowed more efficient viral propagation due to increased cell membrane anchoring of the S protein. The S protein was integrated into rVSV-Venus-SADS SΔ11 particles, susceptible to neutralization by sera from SADS-CoV S1 protein-immunized rabbits. Additionally, we found that TMPRSS2 promotes SADS-CoV spike-mediated cell entry. Furthermore, we assessed the serum-neutralizing ability of mice vaccinated with rVSV-Venus-SADS SΔ11 using a prime-boost immunization strategy, revealing effective neutralizing antibodies against SADS-CoV infection. In conclusion, we have developed a safe and practical tool for studying SADS-CoV entry and exploring the potential of a recombinant VSV-vectored SADS-CoV vaccine.IMPORTANCEZoonotic coronaviruses, like swine acute diarrhea syndrome coronavirus (SADS-CoV), pose a continual threat to human and animal health. To combat this, we engineered a safe and efficient tool by modifying the Vesicular Stomatitis Virus (VSV), creating a replication-competent rVSV-Venus-SADS S virus. Through serial passages, we optimized the virus for enhanced membrane anchoring, a key factor in viral propagation. This modified virus, rVSV-Venus-SADS SΔ11, proved susceptible to neutralization, opening avenues for potential vaccines. Additionally, our study revealed the role of TMPRSS2 in SADS-CoV entry. Mice vaccinated with rVSV-Venus-SADS SΔ11 developed potent neutralizing antibodies against SADS-CoV. In conclusion, our work presents a secure and practical tool for studying SADS-CoV entry and explores the promise of a recombinant VSV-vectored SADS-CoV vaccine.

Protective Efficacy of Lyophilized Vesicular Stomatitis Virus-Based Vaccines in Animal Model.

We evaluated the in vitro effects of lyophilization for 2 vesicular stomatitis virus-based vaccines by using 3 stabilizing formulations and demonstrated protective immunity of lyophilized/reconstituted vaccine in guinea pigs. Lyophilization increased stability of the vaccines, but specific vesicular stomatitis virus-based vaccines will each require extensive analysis to optimize stabilizing formulations.

Mechanisms of active diffusion of vesicular stomatitis virus inclusion bodies and cellular early endosomes in the cytoplasm of mammalian cells.

Viral and cellular particles too large to freely diffuse have two different types of mobility in the eukaryotic cell cytoplasm: directed motion mediated by motor proteins moving along cytoskeletal elements with the particle as its load, and motion in random directions mediated by motor proteins interconnecting cytoskeletal elements. The latter motion is referred to as "active diffusion." Mechanisms of directed motion have been extensively studied compared to mechanisms of active diffusion, despite the observation that active diffusion is more common for many viral and cellular particles. Our previous research showed that active diffusion of vesicular stomatitis virus (VSV) ribonucleoproteins (RNPs) in the cytoplasm consists of hopping between traps and that actin filaments and myosin II motors are components of the hop-trap mechanism. This raises the question whether similar mechanisms mediate random motion of larger particles with different physical and biological properties. Live-cell fluorescence imaging and a variational Bayesian analysis used in pattern recognition and machine learning were used to determine the molecular mechanisms of random motion of VSV inclusion bodies and cellular early endosomes. VSV inclusion bodies are membraneless cellular compartments that are the major sites of viral RNA synthesis, and early endosomes are representative of cellular membrane-bound organelles. Like VSV RNPs, inclusion bodies and early endosomes moved from one trapped state to another, but the distance between states was inconsistent with hopping between traps, indicating that the apparent state-to-state movement is mediated by trap movement. Like VSV RNPs, treatment with the actin filament depolymerizing inhibitor latrunculin A increased VSV inclusion body mobility by increasing the size of the traps. In contrast neither treatment with latrunculin A nor depolymerization of microtubules by nocodazole treatment affected the size of traps that confine early endosome mobility, indicating that intermediate filaments are likely major trap components for these cellular organelles.

Bivalent VSV Vectors Mediate Rapid and Potent Protection from Andes Virus Challenge in Hamsters.

Orthohantaviruses may cause hemorrhagic fever with renal syndrome or hantavirus cardiopulmonary syndrome. Andes virus (ANDV) is the only orthohantavirus associated with human-human transmission. Therefore, emergency vaccination would be a valuable public health measure to combat ANDV-derived infection clusters. Here, we utilized a promising vesicular stomatitis virus (VSV)-based vaccine to advance the approach for emergency applications. We compared monovalent and bivalent VSV vectors containing the Ebola virus (EBOV), glycoprotein (GP), and ANDV glycoprotein precursor (GPC) for protective efficacy in pre-, peri- and post-exposure immunization by the intraperitoneal and intranasal routes. Inclusion of the EBOV GP was based on its favorable immune cell targeting and the strong innate responses elicited by the VSV-EBOV vaccine. Our data indicates no difference of ANDV GPC expressing VSV vectors in pre-exposure immunization independent of route, but a potential benefit of the bivalent VSVs following peri- and post-exposure intraperitoneal vaccination.

Vesicular Stomatitis Virus.

Vesicular stomatitis (VS) is a vector-borne livestock disease caused by either VS New Jersey virus or VS Indiana virus. The disease circulates endemically in northern South America, Central America, and Mexico and only occasionally causes outbreaks in the United States. During the past 20 years, VS outbreaks in the southwestern and Rocky Mountain regions occurred periodically with incursion years followed by virus overwintering and subsequent expansion outbreak years. Regulatory response by animal health officials prevents spread from lesioned animals and manages trade impacts. Recent US outbreaks highlight potential climate change impacts on insect vectors or other transmission-related variables.

Antiviral Effect of Extracellular Matrix Protein ABI3BP on Vesicular Stomatitis Virus and Its Mechanism: A Preliminary Study In Vitro.

Objective To explore the influence of extracellular matrix protein ABI-interactor 3-binding protein (ABI3BP) on vesicular stomatitis virus (VSV) genome replication and innate immune signaling pathway.Methods The small interfering RNA (siRNA) was transfected to knock down ABI3BP gene in human skin fibroblast BJ-5ta cells. VSV-green fluorescent protein (VSV-GFP)-infected cell model was established. The morphological changes and F-actin stress fiber formation were detected on ABI3BP knockdown cells by phalloidin immunofluorescence staining. The mRNA level of virus replication was detected by RT-qPCR in BJ-5ta cells after VSV-GFP infection; western blotting was performed to detect the changes in interferon regulatory factor 3 (IRF3) and TANK-binding kinase 1 (TBK1) phosphorylation levels.Results The VSV-GFP-infected BJ-5ta cell model was successfully established. Efficient knockdown of ABI3BP in BJ-5ta cells was achieved. Phalloidin immunofluorescence staining revealed structural rearrangement of intracellular F-actin after ABI3BP gene knockdown. Compared with the control group, the gene copy number of VSV-GFP in ABI3BP knockdown cells increased by 2.2 - 3.5 times (P<0.01) and 2.2 - 4.0 times (P<0.01) respectively when infected with VSV of multiplicity of infection 0.1 and 1. The expression of viral protein significantly increased in ABI3BP knockdown cells after virus infection. The activation of type-I interferon pathway, as determined by phosphorylated IRF3 and phosphorylated TBK1, was significantly decreased in ABI3BP knockdown cells after VSV-GFP infection.Conclusions Extracellular matrix protein ABI3BP plays an important role in maintaining the formation and rearrangement of actin structure. ABI3BP gene deletion promotes RNA virus replication, and ABI3BP is an important molecule that maintains the integrity of type I interferon pathway.

Stochastic model of vesicular stomatitis virus replication reveals mutational effects on virion production.

We present the first complete stochastic model of vesicular stomatitis virus (VSV) intracellular replication. Previous models developed to capture VSV's intracellular replication have either been ODE-based or have not represented the complete replicative cycle, limiting our ability to understand the impact of the stochastic nature of early cellular infections on virion production between cells and how these dynamics change in response to mutations. Our model accurately predicts changes in mean virion production in gene-shuffled VSV variants and can capture the distribution of the number of viruses produced. This model has allowed us to enhance our understanding of intercellular variability in virion production, which appears to be influenced by the duration of the early phase of infection, and variation between variants, arising from balancing the time the genome spends in the active state, the speed of incorporating new genomes into virions, and the production of viral components. Being a stochastic model, we can also assess other effects of mutations beyond just the mean number of virions produced, including the probability of aborted infections and the standard deviation of the number of virions produced. Our model provides a biologically interpretable framework for studying the stochastic nature of VSV replication, shedding light on the mechanisms underlying variation in virion production. In the future, this model could enable the design of more complex viral phenotypes when attenuating VSV, moving beyond solely considering the mean number of virions produced.

Production of recombinant vesicular stomatitis virus-based vectors by tangential flow depth filtration.

Cell culture-based production of vector-based vaccines and virotherapeutics is of increasing interest. The vectors used not only retain their ability to infect cells but also induce robust immune responses. Using two recombinant vesicular stomatitis virus (rVSV)-based constructs, we performed a proof-of-concept study regarding an integrated closed single-use perfusion system that allows continuous virus harvesting and clarification. Using suspension BHK-21 cells and a fusogenic oncolytic hybrid of vesicular stomatitis virus and Newcastle disease virus (rVSV-NDV), a modified alternating tangential flow device (mATF) or tangential flow depth filtration (TFDF) systems were used for cell retention. As the hollow fibers of the former are characterized by a large internal lumen (0.75 mm; pore size 0.65 µm), membrane blocking by the multi-nucleated syncytia formed during infection could be prevented. However, virus particles were completely retained. In contrast, the TFDF filter unit (lumen 3.15 mm, pore size 2-5 µm) allowed not only to achieve high viable cell concentrations (VCC, 16.4-20.6×106 cells/mL) but also continuous vector harvesting and clarification. Compared to an optimized batch process, 11-fold higher infectious virus titers were obtained in the clarified permeate (maximum 7.5×109 TCID50/mL). Using HEK293-SF cells and a rVSV vector expressing a green fluorescent protein, perfusion cultivations resulted in a maximum VCC of 11.3×106 cells/mL and infectious virus titers up to 7.1×1010 TCID50/mL in the permeate. Not only continuous harvesting but also clarification was possible. Although the cell-specific virus yield decreased relative to a batch process established as a control, an increased space-time yield was obtained. KEY POINTS: • Viral vector production using a TFDF perfusion system resulted in a 460% increase in space-time yield • Use of a TFDF system allowed continuous virus harvesting and clarification • TFDF perfusion system has great potential towards the establishment of an intensified vector production.

Oncolytic Therapy of Solid Tumors by Modified Vesicular Stomatitis Virus.

Vesicular stomatitis virus (VSV) is a promising oncolytic virus for treating solid tumors. We recently engineered a replicating VSV that specifically targets and destroys Her2/neu-expressing cancer cells. This virus was created by eliminating its natural binding site and adding a coding sequence for a single chain antibody to the Her2/neu receptor into its genome. Such an approach can be tailored to target various cellular surface molecules. This mini review will discuss genomic modifications of VSVs and their role in oncolytic therapy and discuss some challenges for moving VSVs to clinical applications.

Adaptation of an rVSV Ebola vaccine purification process for rapid development of a viral vaccine candidate for SARS-CoV-2.

During the COVID-19 pandemic, long development timelines typically associated with vaccines were challenged. The urgent need for a vaccine provided a strong driver to reevaluate existing vaccine development approaches. Innovative approaches to regulatory approval were realized, including the use of platform-based technology. In collaboration with the International AIDS Vaccine Initiative, Inc. (IAVI), Merck & Co., Inc., Rahway, NJ, USA rapidly advanced an investigational SARS-CoV-2 vaccine based on the recombinant vesicular stomatitis virus (rVSV) platform used for the Ebola vaccine ERVEBO (rVSV∆G-ZEBOV-GP). An rVSV∆G-SARS-CoV-2 vaccine candidate was generated using the SARS-CoV-2 spike protein to replace the VSV G protein. The purification process development for this vaccine candidate was detailed in this paper. Areas were highlighted where the ERVEBO platform process was successfully adopted and where additional measures were needed for the SARS-CoV-2 vaccine candidate. These included: (i) endonuclease addition directly into the bioreactor prior to harvest, (ii) inclusion of a core-shell chromatography step for improved purification, and (iii) incorporation of a terminal, sterile filtration step to eliminate the need for aseptic, closed processing. High infectious virus titers were achieved in Phase 3 clinical drug substance (>108 PFU mL-1 ), and process consistency was demonstrated across four large scale batches that were completed in 6 months from clone selection.

Synergistic VSV Virotherapy and Carbon Nanotube Photothermal Therapy for Osteosarcoma in Murine Models.

BACKGROUND/AIM: Wide resection is usually performed for malignant bone and soft tissue tumors, but there is often functional impairment of the affected limb. In this study, we performed virotherapy with the vesicular stomatitis virus (VSV) and photothermal therapy using carbon nanotubes (CNTs) in combination for osteosarcoma, followed by marginal excision. The possibility of local treatment of the primary tumor was then assessed. MATERIALS AND METHODS: LM-8 cells (1×107) were subcutaneously implanted into 5-week-old mice to generate an in vivo osteosarcoma mouse model. Marginectomy was performed. Four groups with six mice each were created: VSV+SWCNTs group, VSV group, SWCNTs group, and an untreated group. Tumor margin resection was performed 2 weeks after tumor cell transplantation. The primary tumor volume, local recurrence, distant metastasis, and survival rate were evaluated. RESULTS: The combination of VSV virotherapy and CNTs photothermal therapy resulted in shrinkage of the primary tumor and reduced local recurrence after marginectomy. There was no significant difference in distant metastasis or survival rate for all groups. CONCLUSION: Combining virotherapy with VSV and CNTs photothermal therapy is useful for local treatment of osteosarcoma in murine models, possibly allowing for smaller tumor resection margins.

Pralatrexate inhibited the replication of varicella zoster virus and vesicular stomatitis virus: An old dog with new tricks.

Varicella zoster virus (VZV) is associated with herpes zoster (HZ) or herpes zoster ophthalmicus (HZO). All antiviral agents currently licensed for the management of VZV replication via modulating different mechanisms, and the resistance is on the rise. There is a need to develop new antiviral agents with distinct mechanisms of action and adequate safety profiles. Pralatrexate (PDX) is a fourth-generation anti-folate agent with an inhibitory activity on folate (FA) metabolism and has been used as an anti-tumor drug. We observed that PDX possessed potent inhibitory activity against VZV infection. In this study, we reported the antiviral effects and the underlying mechanism of PDX against VZV infection. The results showed that PDX not only inhibited VZV replication in vitro and in mice corneal tissues but also reduced the inflammatory response and apoptosis induced by viral infection. Furthermore, PDX treatment showed a similar anti-VSV inhibitory effect in both in vitro and in vivo models. Mechanistically, PDX inhibited viral replication by interrupting the substrate supply for de novo purine and thymidine synthesis. In conclusion, this study discovered the potent antiviral activity of PDX with a novel mechanism and presented a new strategy for VZV treatment that targets a cellular metabolic mechanism essential for viral replication. The present study provided a new insight into the development of broad-spectrum antiviral agents.

Re-Engineered Pseudoviruses for Precise and Robust 3D Mapping of Viral Infection.

Engineered vesicular stomatitis virus (VSV) pseudotyping offers an essential method for exploring virus-cell interactions, particularly for viruses that require high biosafety levels. Although this approach has been employed effectively, the current methodologies for virus visualization and labeling can interfere with infectivity and lead to misinterpretation of results. In this study, we introduce an innovative approach combining genetic code expansion (GCE) and click chemistry with pseudotyped VSV to produce highly fluorescent and infectious pseudoviruses (clickVSVs). These clickVSVs enable robust and precise virus-cell interaction studies without compromising the biological function of the viral surface proteins. We evaluated this approach by generating VSVs bearing a unique chemical handle for click labeling and assessing the infectivity in relevant cell lines. Our results demonstrate that clickVSVs maintain their infectivity post-labeling and present an efficiency about two times higher in detecting surface proteins compared to classical immunolabeling. The utilization of clickVSVs further allowed us to visualize and track 3D virus binding and infection in living cells, offering enhanced observation of virus-host interactions. Thus, clickVSVs provide an efficient alternative for virus-associated research under the standard biosafety levels.