Enteric viruses replicate in salivary glands and infect through saliva.

Enteric viruses like norovirus, rotavirus and astrovirus have long been accepted as spreading in the population through fecal-oral transmission: viruses are shed into feces from one host and enter the oral cavity of another, bypassing salivary glands (SGs) and reaching the intestines to replicate, be shed in feces and repeat the transmission cycle1. Yet there are viruses (for example, rabies) that infect the SGs2,3, making the oral cavity one site of replication and saliva one conduit of transmission. Here we report that enteric viruses productively and persistently infect SGs, reaching titres comparable to those in the intestines. We demonstrate that enteric viruses get released into the saliva, identifying a second route of viral transmission. This is particularly significant for infected infants, whose saliva directly transmits enteric viruses to their mothers' mammary glands through backflow during suckling. This sidesteps the conventional gut-mammary axis route4 and leads to a rapid surge in maternal milk secretory IgA antibodies5,6. Lastly, we show that SG-derived spheroids7 and cell lines8 can replicate and propagate enteric viruses, generating a scalable and manageable system of production. Collectively, our research uncovers a new transmission route for enteric viruses with implications for therapeutics, diagnostics and importantly sanitation measures to prevent spread through saliva.

Cost-effective 3D lung tissue spheroid as a model for SARS-CoV-2 infection and drug screening.

BACKGROUND: The SARS-CoV-2 pandemic has spurred an unparalleled scientific endeavor to elucidate the virus' structure, infection mechanisms, and pathogenesis. Two-dimensional culture systems have been instrumental in shedding light on numerous aspects of COVID-19. However, these in vitro systems lack the physiological complexity to comprehend the infection process and explore treatment options. Three-dimensional (3D) models have been proposed to fill the gap between 2D cultures and in vivo studies. Specifically, spheroids, composed of lung cell types, have been suggested for studying SARS-CoV-2 infection and serving as a drug screening platform. METHODS: 3D lung spheroids were prepared by coculturing human alveolar or bronchial epithelial cells with human lung stromal cells. The morphology, size, and ultrastructure of spheroids before and after SARS-CoV-2 infection were analyzed using optical and electron microscopy. Immunohistochemistry was used to detect spike protein and, thus, the virus presence in the spheroids. Multiplex analysis elucidated the cytokine release after virus infection. RESULTS: The spheroids were stable and kept their size and morphology after SARS-CoV-2 infection despite the presence of multivesicular bodies, endoplasmic reticulum rearrangement, tubular compartment-enclosed vesicles, and the accumulation of viral particles. The spheroid responded to the infection releasing IL-6 and IL-8 cytokines. CONCLUSION: This study demonstrates that coculture spheroids of epithelial and stromal cells can serve as a cost-effective infection model for the SARS-CoV-2 virus. We suggest using this 3D spheroid as a drug screening platform to explore new treatments related to the cytokines released during virus infection, especially for long COVID treatment.

Human Kidney Spheroids and Monolayers Provide Insights into SARS-CoV-2 Renal Interactions.

BACKGROUND: Although coronavirus disease 2019 (COVID-19) causes significan t morbidity, mainly from pulmonary involvement, extrapulmonary symptoms are also major componen ts of the disease. Kidney disease, usually presenting as AKI, is particularly severe among patients with COVID-19. It is unknown, however, whether such injury results from direct kidney infection with COVID-19's causative virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), or from indirect mechanisms. METHODS: Using ex vivo cell models, we sought to analyze SARS-CoV-2 interactions with kidney tubular cells and assess direct tubular injury. These models comprised primary human kidney epithelial cells (derived from nephrectomies) and grown as either proliferating monolayers or quiescent three-dimensional kidney spheroids. RESULTS: We demonstrated that viral entry molecules and high baseline levels of type 1 IFN-related molecules were present in monolayers and kidney spheroids. Although both models support viral infection and replication, they did not exhibit a cytopathic effect and cell death, outcomes that were strongly present in SARS-CoV-2-infected controls (African green monkey kidney clone E6 [Vero E6] cultures). A comparison of monolayer and spheroid cultures demonstrated higher infectivity and replication of SARS-CoV-2 in actively proliferating monolayers, although the spheroid cultures exhibited high er levels of ACE2. Monolayers exhibited elevation of some tubular injury molecules-including molecules related to fibrosis (COL1A1 and STAT6) and dedifferentiation (SNAI2)-and a loss of cell identity, evident by reduction in megalin (LRP2). The three-dimensional spheroids were less prone to such injury. CONCLUSIONS: SARS-CoV-2 can infect kidney cells without a cytopathic effect. AKI-induced cellular proliferation may potentially intensify infectivity and tubular damage by SARS-CoV-2, suggesting that early intervention in AKI is warranted to help minimize kidney infection.

Spheroids and organoids as humanized 3D scaffold-free engineered tissues for SARS-CoV-2 viral infection and drug screening.

The new coronavirus (2019-nCoV) or the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was officially declared by the World Health Organization (WHO) as a pandemic in March 2020. To date, there are no specific antiviral drugs proven to be effective in treating SARS-CoV-2, requiring joint efforts from different research fronts to discover the best route of treatment. The first decisions in drug discovery are based on 2D cell culture using high-throughput screening. In this context, spheroids and organoids emerge as a reliable alternative. Both are scaffold-free 3D engineered constructs that recapitulate key cellular and molecular events of tissue physiology. Different studies have already shown their advantages as a model for different infectious diseases, including SARS-CoV-2 and for drug screening. The use of these 3D engineered tissues as an in vitro model can fill the gap between 2D cell culture and in vivo preclinical assays (animal models) as they could recapitulate the entire viral life cycle. The main objective of this review is to understand spheroid and organoid biology, highlighting their advantages and disadvantages, and how these scaffold-free engineered tissues can contribute to a better comprehension of viral infection by SARS-CoV-2 and to the development of in vitro high-throughput models for drug screening.

Herpes Simplex Virus 1 Infection Promotes the Growth of a Subpopulation of Tumor Cells in Three-Dimensional Uveal Melanoma Cultures.

Herpes simplex virus 1 (HSV-1)-mediated oncolytic therapy is an emerging cancer treatment modality with potential effectiveness against a variety of malignancies. To better understand the interaction of HSV-1 with neoplastic cells, we inoculated three-dimensional (3D) cultures of human uveal melanoma cells with HSV-1. 3D melanoma cultures were established by placing tumor cells on the surface of a Matrigel matrix, which was followed by the growth of tumor cells on the matrix surface and invasion of the Matrigel matrix by some tumor cells to form multicellular tumor spheroids within the matrix. When established 3D melanoma cultures were inoculated with HSV-1 by placing virus on the surface of cultures, virus infection caused extensive death of melanoma cells growing on the surface of the 3D matrix and significantly decreased the number of tumor cell spheroids within the matrix. However, HSV-1 infection did not lead to a complete destruction of tumor cells in the 3D cultures during a 17-day observation period and, surprisingly, HSV-1 infection promoted the growth of some melanoma cells within the matrix as determined by the significantly increased size of residual viable multicellular tumor spheroids in virus-inoculated 3D cultures at 17 days after virus inoculation. Acyclovir treatment inhibited HSV-1-induced tumor cell killing but did not block the virus infection-induced increase in spheroid size. These findings suggest that although HSV-1 oncolytic virotherapy may cause extensive tumor cell killing, it may also be associated with the unintended promotion of the growth of some tumor cells.IMPORTANCE Cancer cells are exposed to HSV-1 during oncolytic virotherapy with the intention of killing tumor cells. Our observations reported here suggest that potential dangers of HSV-1 oncolytic therapy include promotion of growth of some tumor cells. Furthermore, our findings raise the possibility that HSV-1 infection of neoplastic cells during natural infections or vaccinations may promote the growth of tumors. Our study indicates that HSV-1 infection of 3D tumor cell cultures provides an experimental platform in which mechanisms of HSV-1-mediated promotion of tumor cell growth can be effectively studied.

Stem Cell Properties of Normal Human Keratinocytes Determine Transformation Responses to Human Papillomavirus 16 DNA.

Human papillomavirus (HPV) infection of the genital tract is common; however, only about 10 to 15% of infections persist, and approximately 10 to 15% of these persistent infections result in cancer. Basal epidermal stem cells are the presumed target cells for HPV infection, providing a reservoir of latently infected cells that persist over time and initiate lesions. However, it is not known whether stem cell density has any influence on transformation of human keratinocytes by HPV. We explored the relationship between stem cell properties of normal human keratinocytes and their susceptibility to transformation by HPV16 DNA. Normal human keratinocyte isolates (NHKc) derived from different donors were cultured in three-dimensional anchorage-free suspension to assess their spheroid-forming ability. NHKc spheroids were then plated back into plastic monolayer culture and transfected with full-length HPV16 DNA, which we have previously shown to integrate into the host cell genome upon transfection. Spheroid-derived NHKc (SD-NHKc) and fluorescence-activated cell sorting-purified populations of basal stem-like keratinocytes, expressing low levels of epidermal growth factor receptor and high levels of integrin alpha 6 (EGFRlo/ITGα6hi), responded to transfection with HPV16 DNA with more vigorous proliferation, greater immortalization efficiency, and faster progression to differentiation resistance than autologous mass-cultured cells. Conversely, cells committed to terminal differentiation (EGFRhi/ITGα6lo) grew slowly after transfection with HPV16 and failed to generate immortalized or DR clones. HPV16 DNA induced stem cell properties in mass-cultured NHKc. We conclude that HPV16 preferentially immortalizes basal keratinocytes with stem cell properties and that these cells readily achieve a differentiation-resistant phenotype upon immortalization by HPV16.IMPORTANCE This paper explores the relationship between the stem cell properties of normal human epidermal cells in culture and these cells' susceptibility to transformation by HPV16 DNA, the HPV type present in about 50% of cervical cancers. We report variable susceptibilities to HPV16-mediated transformation among different keratinocyte isolates derived from neonatal foreskin. Our findings provide strong experimental evidence that HPV16 preferentially transforms basal keratinocytes with stem cell properties. Insights gained from these studies increase our understanding of the host cell-specific factors influencing individual susceptibility to HPV-driven transformation and the contributing factors leading to preneoplastic and neoplastic progression of HPV-positive lesions.

MicroRNA-183 suppresses cancer stem-like cell properties in EBV-associated nasopharyngeal carcinoma.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV)-associated epithelial malignancy that exhibits distinct geographical and ethnic prevalence. Although the contemporary therapeutic approach of radio-/chemotherapy provides excellent results for patients with early-stage disease, it is far from satisfactory for those with disease remission and distant metastasis. Promising therapeutic strategies for advanced and relapsed NPC are still lacking. We recently identified and characterized a cancer stem-like cell (CSC) subpopulation in NPC that appeared to play an important role in tumor progression. Microarray analysis revealed downregulation of several stemness-inhibiting miRNAs in these CSC cells. Among these miRNAs, miR-96 and miR-183 showed the highest fold change and were selected to elucidate their role in repressing NPC CSC properties. METHODS: MiR-96 and miR-183 expression in NPC CSCs was detected by qRT-PCR. Transient and stable transfection was performed in EBV-positive NPC C666-1 cells to examine the effects of ectopic expression of miR-96 and miR-183 on repressing cell growth and CSC properties. Anchorage-dependent (colony formation) and anchorage-independent (tumor sphere formation) growths of these miR-96 and miR-183 expressing cells were determined. Expression of multiple CSC markers and related molecules were accessed by flow cytometry and Western blotting. The tumorigenicity of the stable miR-96- and miR-183-transfected NPC cells was examined in an in vivo nude mice model. RESULTS: Downregulation of miR-96 and miR-183 was confirmed in NPC spheroids. Using transient or stable transfection, we showed that ectopic expression of miR-96 and miR-183 suppressed cell growth and tumor sphere formation in NPC. Reduced NICD3 and NICD4 in miR-96- and miR-183-expressing NPC cells suggests the involvement of the NOTCH signaling pathway in their tumor suppressive function. Finally, we showed that the tumorigenicity of cells stably expressing miR-183 was significantly inhibited in the in vivo nude mice model. CONCLUSIONS: miR-183 is a tumor-suppressive miRNA in EBV-associated NPC. Its abilities to suppress CSC properties in vitro and effectively reduce tumor growth in vivo shed light on its role as a potential therapeutic target.

Mammalian orthoreovirus T3D infects U-118 MG cell spheroids independent of junction adhesion molecule-A.

In the canonical pathway, infection of cells by the wild-type mammalian orthoreovirus Type 3 Dearing (T3D) is dependent on the interaction of the viral spike protein σ1 with the high-affinity cellular receptor junction adhesion molecule-A (JAM-A). We previously demonstrated that the human glioblastoma cell line U-118 MG does not express JAM-A and resists reovirus T3D infection in standard cell culture conditions (SCCC). Heterologous JAM-A expression sensitises U-118 MG cells to reovirus T3D. Here we studied reovirus infection in U-118 MG cells grown in spheroid cultures with the premise that cells in such cultures resemble cells in tumours more than those grown under standard adherent cell culture conditions on a plastic surface. Although the U-118 MG cells in spheroids do not express JAM-A, they are susceptible to reovirus T3D infection. We show that this can be attributed to factors secreted by cells in the spheroids. The concentration of active extracellular proteases cathepsin B and L in the medium of spheroid cultures was increased 19- and 24-fold, respectively, as compared with SCCC. These enzymes can convert the reovirus particles into a form that can infect the U-118 MG cells independent of JAM-A. Taken together, these data demonstrate that infection of tumour cells by wild-type reovirus T3D is not strictly dependent on the expression of JAM-A on the cell surface.

Scalable spheroid model of human hepatocytes for hepatitis C infection and replication.

Developing effective new drugs against hepatitis C (HCV) virus has been challenging due to the lack of appropriate small animal and in vitro models recapitulating the entire life cycle of the virus. Current in vitro models fail to recapitulate the complexity of human liver physiology. Here we present a method to study HCV infection and replication on spheroid cultures of Huh 7.5 cells and primary human hepatocytes. Spheroid cultures are constructed using a galactosylated cellulosic sponge with homogeneous macroporosity, enabling the formation and maintenance of uniformly sized spheroids. This facilitates easy handling of the tissue-engineered constructs and overcomes limitations inherent of traditional spheroid cultures. Spheroids formed in the galactosylated cellulosic sponge show enhanced hepatic functions in Huh 7.5 cells and maintain liver-specific functions of primary human hepatocytes for 2 weeks in culture. Establishment of apical and basolateral polarity along with the expression and localization of all HCV specific entry proteins allow for a 9-fold increase in viral entry in spheroid cultures over conventional monolayer cultures. Huh 7.5 cells cultured in the galactosylated cellulosic sponge also support replication of the HCV clone, JFH (Japanese fulminant hepatitis)-1 at higher levels than in monolayer cultures. The advantages of our system in maintaining liver-specific functions and allowing HCV infection together with its ease of handling make it suitable for the study of HCV biology in basic research and pharmaceutical R&D.

Multifaceted cancer alleviation by cowpea mosaic virus in a bioprinted ovarian cancer peritoneal spheroid model.

Ovarian cancer (OvCa) is a leading cause of mortality among gynecological malignancies and usually manifests as intraperitoneal spheroids that generate metastases, ascites, and an immunosuppressive tumor microenvironment. In this study, we explore the immunomodulatory properties of cowpea mosaic virus (CPMV) as an adjuvant immunotherapeutic agent using an in vitro model of OvCa peritoneal spheroids. Previous findings highlighted the potent efficacy of intratumoral CPMV against OvCa in mouse tumor models. Leveraging the precision control over material deposition and cell patterning afforded by digital-light-processing (DLP) based bioprinting, we constructed OvCa-macrophage spheroids to mimic peritoneal spheroids using gelatin methacrylate (GelMA), a collagen-derived photopolymerizable biomaterial to mimic the extracellular matrix. Following CPMV treatment, bioprinted spheroids exhibited inhibited OvCa progression mediated by macrophage activation. Our analysis indicates that CPMV regulates and activates macrophage to both induce OvCa cell killing and restore normal cell-cell junctions. This study deepened our understanding of the mechanism of CPMV intratumoral immunotherapy in the setting of OvCa. This study also highlights the potential of studying immunotherapies using high throughput tissue models via DLP bioprinting.

3D Spheroid and Organoid Models to Study Neuroinfection of RNA Viruses.

Three-dimensional culture models of the brain enable the study of neuroinfection in the context of a complex interconnected cell matrix. Depending on the differentiation status of the neural cells, two models exist: 3D spheroids also called neurospheres and cerebral organoids. Here, we describe the preparation of 3D spheroids and cerebral organoids and give an outlook on their usage to study Rift Valley fever virus and other neurotropic viruses.

Imaging herpes simplex virus type 1 amplicon vector-mediated gene expression in human glioma spheroids.

Vectors derived from herpes simplex virus type 1 (HSV-1) have great potential for transducing therapeutic genes into the central nervous system; however, inefficient distribution of vector particles in vivo may limit their therapeutic potential in patients with gliomas. This study was performed to investigate the extent of HSV-1 amplicon vector-mediated gene expression in a three-dimensional glioma model of multicellular spheroids by imaging highly infectious HSV-1 virions expressing green fluorescent protein (HSV-GFP). After infection or microscopy-guided vector injection of glioma spheroids at various spheroid sizes, injection pressures and injection times, the extent of HSV-1 vector-mediated gene expression was investigated via laser scanning microscopy. Infection of spheroids with HSV-GFP demonstrated a maximal depth of vector-mediated GFP expression at 70 to 80 µm. A > 80% transduction efficiency was reached only in small spheroids with a diameter of < 150 µm. Guided vector injection into the spheroids showed transduction efficiencies ranging between < 10 and > 90%. The results demonstrated that vector-mediated gene expression in glioma spheroids was strongly dependent on the mode of vector application-injection pressure and injection time being the most important parameters. The assessment of these vector application parameters in tissue models will contribute to the development of safe and efficient gene therapy protocols for clinical application.

3D culture conditions support Kaposi's sarcoma herpesvirus (KSHV) maintenance and viral spread in endothelial cells.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a human tumorigenic virus and the etiological agent of an endothelial tumor (Kaposi's sarcoma) and two B cell proliferative diseases (primary effusion lymphoma and multicentric Castleman's disease). While in patients with late stage of Kaposi's sarcoma the majority of spindle cells are KSHV-infected, viral copies are rapidly lost in vitro, both upon culture of tumor-derived cells or from newly infected endothelial cells. We addressed this discrepancy by investigating a KSHV-infected endothelial cell line in various culture conditions and in tumors of xenografted mice. We show that, in contrast to two-dimensional endothelial cell cultures, KSHV genomes are maintained under 3D cell culture conditions and in vivo. Additionally, an increased rate of newly infected cells was detected in 3D cell culture. Furthermore, we show that the PI3K/Akt/mTOR and ATM/γH2AX pathways are modulated and support an improved KSHV persistence in 3D cell culture. These mechanisms may contribute to the persistence of KSHV in tumor tissue in vivo and provide a novel target for KS specific therapeutic interventions. KEY MESSAGES: In vivo maintenance of episomal KSHV can be mimicked in 3D spheroid cultures 3D maintenance of KSHV is associated with an increased de novo infection frequency PI3K/Akt/mTOR and ATM/ γH2AX pathways contribute to viral maintenance.

Evaluation of Bystander Infection of Oncolytic Virus using a Medium Flow Integrated 3D In Vitro Microphysiological System.

Replicable oncolytic viruses (OVs) induce tumor cell lysis and release viral progeny. The released progeny virions and cell debris can spread within surrounding tumor cells or blood vessels. These released molecules may also induce bystander damage in additional tumor cells through spreading within surrounding tumor cells or blood vessels. However, this effect has not been clearly demonstrated due to the difficulty of direct observation. Here, the bystander infection of OVs by vessel delivery and selective infection in 3D multicellular tumoroids (MCTs) in an in vitro microphysiological system (MPS) with integrated medium flow is demonstrated. This study uses replicable vesicular stomatitis virus (VSV)-green fluorescence protein (GFP) to identify the location of infection in 3D MCTs. Using this MPS, the oncoselective infection by VSV-GFP and the spreading by delivery of OVs through flow via block-to-block linkage of the primary infected MPS with uninfected 3D MCTs in an integrated MPS is observed. This MPS enables real-time monitoring and various analysis for the bystander infection of OVs. It is expected that the 3D in vitro MPS can be suitable to investigate the oncoselective spreading and bystander infection of OVs.

Mechanism of baricitinib supports artificial intelligence-predicted testing in COVID-19 patients.

Baricitinib is an oral Janus kinase (JAK)1/JAK2 inhibitor approved for the treatment of rheumatoid arthritis (RA) that was independently predicted, using artificial intelligence (AI) algorithms, to be useful for COVID-19 infection via proposed anti-cytokine effects and as an inhibitor of host cell viral propagation. We evaluated the in vitro pharmacology of baricitinib across relevant leukocyte subpopulations coupled to its in vivo pharmacokinetics and showed it inhibited signaling of cytokines implicated in COVID-19 infection. We validated the AI-predicted biochemical inhibitory effects of baricitinib on human numb-associated kinase (hNAK) members measuring nanomolar affinities for AAK1, BIKE, and GAK. Inhibition of NAKs led to reduced viral infectivity with baricitinib using human primary liver spheroids. These effects occurred at exposure levels seen clinically. In a case series of patients with bilateral COVID-19 pneumonia, baricitinib treatment was associated with clinical and radiologic recovery, a rapid decline in SARS-CoV-2 viral load, inflammatory markers, and IL-6 levels. Collectively, these data support further evaluation of the anti-cytokine and anti-viral activity of baricitinib and support its assessment in randomized trials in hospitalized COVID-19 patients.

Enhanced in vitro virus expression using 3-dimensional cell culture spheroids for infection.

The way viruses interact with cultured cells and their surrounding environment is still a matter of debate. From a technical point of view, 2D cell cultures only partially exhibit the morpho-molecular pattern required for viral tropism, not reflecting the complexity of the microenvironment in vivo. Therefore, 3D cell cultures are envisioned as an alternative approach to study viral replication possibly closer to in vivo conditions than 2D, representing the link between traditional cell culture and in vivo models. The use of cellular spheroids is proving to be useful to optimize and overcome constraints related to conventional in vitro systems for viral isolation. In order to create an advanced 3D in vitro isolation system, we compared the classic 2D shell vial system with the spheroid culture method based on the adhesion inhibition technique with pHema. In this study, we evaluated which of the most common viral cell lines used in our laboratory (A-549, 293 T, CaCo2, KB, HUH-7, VERO, and MRC-5) (Fig. 1) could be grown as 3D cultures and all proved to be able to grow as spheroids. Subsequently, we compared the sensitivity and efficiency of isolation of three viral species of medical interest (Adenovirus, CMV, HSV-1) in 2D and 3D cell cultures obtained from the respective susceptible cells. Our results indicate earlier and more sensitive virus isolation than in traditional 2D shell vial system for all three viruses tested, thus confirming how the establishment of 3D culture systems in the virological field is crucial to the improvement and evolution of more accurate and faster virus isolation protocols.

SPLUNC1 and MLL3 regulate cancer stem cells in nasopharyngeal carcinoma.

PURPOSE: Nasopharyngeal carcinoma (NPC) is one of the common types of cancer that originate from the nasopharyngeal region. Recurrence and early metastasis represent major problems associated with NPC mortality. These are mainly caused by various molecular changes that take place during the conversion of normal stem cells into treatment-resistant stem cells. The aim of our study was to investigate the proliferative behavior of cancer stem cells in different stages of NPC and to identify the functional roles of SPLUNC1 and MLL3 associated with cancer stem cells. METHODS: We successfully developed a NPC mouse model using C666-1 cells. Immunohistochemistry and Western blotting were used to analyze the expression of SOX2, SPLUNC1 and MLL3. RESULTS: Null BALB/c mice developed initial and aggressive stages of NPC in 3 and 10 weeks, respectively. Histological results showed that the proliferative ability of cells increased as the tumor progressed to the next level. The SOX2 protein showed a peculiar pattern of upregulation in aggressive NPC when compared with control tissues and initial NPC. Remarkably, our study found that SPLUNC1 and MLL3 expression showed upregulation in initial NPC, which indicates their role in the tumor resistance mechanism even if their expression was downregulated in aggressive NPC. CONCLUSIONS: Our results conclude that SPLUNC1 and MLL3 expression control the resistance mechanism of cancer stem cells in initial NPC, but their downregulation in aggressive stages contributes to developing resistance in nasopharyngeal cancer stem cells.

Numerical modeling of molecular-motor-assisted transport of adenoviral vectors in a spherical cell.

Viral gene delivery in a spherical cell is investigated numerically. The model of intracellular trafficking of adenoviruses is based on molecular-motor-assisted transport equations suggested by Smith and Simmons. These equations are presented in spherical coordinates and extended by accounting for the random component of motion of viral particles bound to filaments. This random component is associated with the stochastic nature of molecular motors responsible for locomotion of viral particles bound to filaments. The equations are solved numerically to simulate viral transport between the cell membrane and cell nucleus during initial stages of viral infection.

STAG2 deficiency induces interferon responses via cGAS-STING pathway and restricts virus infection.

Cohesin is a multi-subunit nuclear protein complex that coordinates sister chromatid separation during cell division. Highly frequent somatic mutations in genes encoding core cohesin subunits have been reported in multiple cancer types. Here, using a genome-wide CRISPR-Cas9 screening approach to identify host dependency factors and novel innate immune regulators of rotavirus (RV) infection, we demonstrate that the loss of STAG2, an important component of the cohesin complex, confers resistance to RV replication in cell culture and human intestinal enteroids. Mechanistically, STAG2 deficiency results in spontaneous genomic DNA damage and robust interferon (IFN) expression via the cGAS-STING cytosolic DNA-sensing pathway. The resultant activation of JAK-STAT signaling and IFN-stimulated gene (ISG) expression broadly protects against virus infections, including RVs. Our work highlights a previously undocumented role of the cohesin complex in regulating IFN homeostasis and identifies new therapeutic avenues for manipulating the innate immunity.

A three-dimensional assay for measurement of viral-induced oncolysis.

Oncolytic viruses represent a novel cancer treatment strategy. Despite their promising preclinical data, however, corresponding clinical trials have disappointed. To aid preclinical analyses, we hypothesized that three-dimensional tumor cell clusters or spheroids might provide an assay system superior to conventional monolayer cell cultures. Spheroids show viral infection, replication and oncolytic patterns distinct from conventional monolayer assays. Therefore, viral tumor penetration and oncolysis measurements may be improved with such three-dimensional models. Also, preclinical analyses of oncolytic viruses frequently measure mitochondrial activity, but more accurate measures of oncolysis might involve quantitation of intracellular protein release. Therefore, we measured luciferase released from luciferase-expressing spheroids and found unique patterns that maintained consistency with various viruses and doses. The relative variations between viruses and doses may represent temporal differences in oncolysis dynamics. Analysis of five recombinant replicative adenoviruses with promise for clinical application showed that Ad5/3-Delta24 produced the most luciferase release 1 week after infection and achieved the earliest and highest peak luciferase release level. Ad5/3-Delta24 also effected the earliest subtotal spheroid cell death. These findings closely parallel monolayer oncolysis assays with these agents. Therefore, the luciferase-expressing tumor spheroid assay represents a promising three-dimensional model for preclinical analysis of replicative oncolytic agents.