Viruses of Avian Origin for Cancer Virotherapy

Viruses of Avian Origin for Cancer Virotherapy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Viruses of Avian Origin for Cancer Virotherapy Jacob Pitcovski Pitcovski, Gilad Gallili, Daria Oren Aharon, Shir Malka, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6690670/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Feb, 2026 Read the published version in Archives of Virology → Version 1 posted 5 You are reading this latest preprint version Abstract Oncolytic viruses (OVs) can selectively infect and kill tumor cells. Although showing promise, several challenges impede OVs broad application in cancer therapy. A major obstacle is limited treatment duration due to preexisting or the induction of neutralizing immune responses toward the OVs following treatment. Widening the reservoir of OVs will allow replacement of treatment viruses following neutralization. This study aimed to identify new OVs and to test their oncolytic effect. Eight avian viruses (AVs) were used to infect human and mice normal and cancerous cell lines, of which three displayed superior ability to selectively kill cancer cells in vitro. These AVs induced cytopathic effects and inhibited proliferation of fifteen out of eighteen cancer cell lines tested, each affecting 5–14 of the cell lines; none affected normal fibroblasts. In vivo , growth of G361 melanoma cell tumors in nude mice was inhibited following intra-tumoral (i.t) injection of AVs. In two models of immunocompetent mice carrying tumors and injected i.t with AVs, tumors growth was significantly delayed. Albeit tumor growth commenced in correlation with the development of anti-virus antibody levels. These tested AVs together with their field-characterized variants, comprise a vast arsenal of potential OVs that may open the possibility of administration of several viruses in mix or in sequence to overcome both tumor resistance due to acquired mutations as well as neutralization by the rise of the acquired immune response. In conclusion, the AVs tested in this study demonstrated OVs characteristics and may be used to enable prolonged cancer virotherapy treatment. Oncolytic viruses Cancer virotherapy Avian reovirus Infectious bursal disease virus Avian metapneumovirus Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1 Introduction An oncolytic virus (OV) is defined by its ability to selectively propagate within and suppress tumor cells without affecting normal cells [1]. Several OVs have been tested in clinical studies and appear to be safe at feasible doses [2]. Clinical studies have demonstrated that OVs may significantly improve the prognosis of cancer patients [3]. However, oncolytic virotherapy is still limited by, for example, neutralizing antibodies raised against the therapeutic OV [4]. This phenomenon, as well as others, necessitates new routes for identification and manufacture of OVs for virotherapy. The mechanisms by which OV differentiate between normal and cancerous cells is known for some of the viruses, while others are not yet resolved. One well characterized mechanism of RNA-virus selectivity for cancer cells is over-activation of the Ras oncogene leading to under-phosphorylation and subsequent inactivation of the RNA-activated protein kinase (PKR) [5]. In normal cells, PKR acts as both a sensor and an effector in the response to viral infections [6]. After sensing double-stranded RNA molecules in the cytoplasm of infected cells, phosphorylated PKR inhibits mRNA translation, triggers apoptosis, and amplifies the IFN response, resulting in inhibition of virus replication [6].This mechanism of selectivity presents an appealing opportunity for use of RNA viruses which are non-pathogenic or infectious in humans as therapeutic OVs. OV can be classified as genetically engineered (e.g., Talimogene laherperepvec (T-VEC)) or natural (e.g., reovirus) viruses. Reovirus type 3 isolated from humans induces direct lysis of cancer cells, as well as antitumor immune activation of dendritic cells, natural killer (NK) cells, and effector T-cells [7]. On this basis, a human reovirus treatment, Reolysin, was developed and tested in multiple clinical trials [5] as monotherapy or in combination with other drugs to treat several cancers, including metastatic breast cancer, advanced-stage head and neck cancer, metastatic ovarian cancer, malignant gliomas, prostate cancer and metastatic melanoma [5,8]. Another natural OV is the Newcastle disease virus (NDV), an avian pathogen which is a member of the paramyxovirus family. The oncolytic potential of both virulent and attenuated NDV strains has been demonstrated in cell cultures, in experimental animal models, and in clinical trials. Preclinical and clinical experience with oncolytic NDV has indicated its potential efficacy for treatment of a variety of lymphomas and solid tumors, including metastases [9]. Freeman et al. reported on the potential efficacy of NDV as a treatment for glioblastoma [10]. As with reovirus, both a direct cytopathic effect and an indirect effect through induction of the immune system were demonstrated for NDV [9]. The current work aimed to evaluate avian metapneumoviruses (aMPV), avian reovirus (ARV), infectious bursal disease virus (IBDV) and infectious bronchitis virus (IBV) for their ability to serve as OVs. The four avian RNA viruses are pathogenic in poultry, but with no documented pathogenicity in humans. aMPV, a member of the Paramyxoviridae family, is an enveloped virus that carries a single-stranded, negative-sense RNA genome [11]. ARV, a member of the Reoviridae family, is a non-enveloped virus with 10 segments of a double-stranded RNA (dsRNA) genome [12]. ARV variants may be divided into four immunogenically distinct genotype clusters which can further extend the OVs arsenal [13]. IBDV, a member of the Birnaviridae family, is a nonenveloped virus that carries two dsRNA segments [14,15]. IBV, a member of the Coronaviridae family, is an enveloped positive-strand RNA virus [11]. 2 MATERIALS AND METHODS 2.1 Cells The cell lines VERO (Green monkey epithelial kidney), SK-BR-3, MCF-7, MDA-KB2, MDA-MB231(human breast cancer), G361 (human melanoma), DU145, PC3 (human prostate cancer), SW480 (human colon cancer), B16-F10 (mouse melanoma), 4T1 (mouse mammary cancer), LLC1 (mouse Lewis lung cancer) and RENCA (mouse renal cancer) were obtained from the American Type Culture Collection (ATCC, USA). Cell lines 697, REH (acute lymphoblastic leukemia [ALL]) and LNCAP (human prostate cancer) were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Germany). Human foreskin fibroblasts primary tissue culture (FF) was kindly provided by Prof. Raphael Gorodetsky of the Sharett Institute Jerusalem. Melanoma lines M-15, M-373, M-374 and M-376 were a kind gift from Prof. Michal Lotem at the Sharett Institute of Oncology, Hadassah Medical Center, Jerusalem. MC-38 (mouse colon cancer) was obtained from the Technion, Israel. FF cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) 4.5 g/l glucose supplemented with 10% fetal calf serum (FCS), 1% L-glutamate, 1% sodium pyruvate 1% MEM non-essential amino acids, 1% MEM vitamin solution and 1% penicillin-streptomycin solution (Biological Industries, Israel). M-15, M-373, M-374 and M-376 cells were grown in DMEM + RPMI (1:1) supplemented with 10% FCS, 1% L-glutamate, 1% sodium pyruvate and 1% penicillin-streptomycin solution. MC-38 cells were grown in DMEM supplemented with 10% FCS, 1% L-glutamate, 1% sodium pyruvate, 1% MEM non-essential amino acids, 1% HEPES and 1% penicillin-streptomycin solution. All other cell lines were maintained under conditions and in the media recommended by ATCC or DSMZ. Cells were incubated at 37°C and in 5% CO 2 . 2.2 Viruses tested 2.2.1 Avian reoviruses (ARV) Reovirus field isolate 5215 was collected from the tendon of infected birds. The tendon fluid was used to infect (100 µl) specific-pathogen-free embryos, and the virus was isolated by mixing 1:1 (v/v) of the allantoic fluid with lysis buffer (10 mM Tris pH 8.0, 10 mM CaCl 2 , 100 mM NaCl), freeze-thawing three times, and centrifuging (10,000 × g , 4°C). Viral presence was determined as described previously by Goldenberg et al. [32]. Briefly, RNA was extracted and concentrated from the supernatant then reverse transcribed and sequenced. The supernatant was stored at 4°C until use. Reovirus s1133, an attenuated vaccine strain, was supplied by Phibro Vaccines (Israel) and propagated in VERO cells. 2.2.2 Infectious bursal disease virus (IBDV) IBDV KS is an Israeli-isolated pathogenic strain, previously characterized and sequenced by Pitcovski et al [12]. IBDV MB is an attenuated vaccine strain, derived from the KS strain [13] and manufactured by Phibro Vaccines (Israel). IBDV Winterfield (IBDV WF; Solvay, Belgium) is an attenuated mild vaccine strain [14], and was propagated in VERO cells. 2.2.3 Avian metapneumovirus (aMVP) aMPV P20 is an attenuated vaccine strain (supplied by Phibro Vaccines, Israel) and was propagated in VERO cells. 2.2.4 Infectious bronchitis virus (IBV) IBV M41 a virulent strain and IBV H120 is a live attenuated vaccine derived from strain M41 (supplied by Phibro Vaccines, Israel). Both were propagated in embryonated eggs and quantified by the supplier. 2.2.5 Preparation of virus stock banks Viruses received lyophilized were suspended according to the manufacturer’s instructions. All liquid virus solutions were aliquoted and stored at -80°C. 2.3 Virus propagation in VERO cells VERO cells were grown to 90–100% confluence in 175 cc 2 flasks. Growth medium was removed, and adherent cells were added with 5 ml of either ARV s1133, IBDV WF or aMPV-P20. Before adding to the cells, the ARV s1133, IBDV WF or aMPV-P20 [at tissue culture infectious dose 50 (TCID 50 )/ml of 8, 7.32 and 5, respectively] were diluted 5 folds in cell infection medium (growth medium with FCS reduced to 0.5%). After a 1-hour incubation, 39 ml cell infection medium were added to flasks and cells were further incubated. On days 4 (ARV) or 8 (IBDV and aMPV), the cytopathic effect was verified under the microscope. Flasks were frozen (-80°C) and thawed (37°C) twice, after which, all content was collected and centrifuged (9800 × g , 10 min, 4°C for ARV s1133 and IBDV WF or 4000 × g , 15 min, 4°C for aMPV). The supernatant (sup) was collected and stored at -80°C. 2.4 Enrichment and concentration of propagated viruses 2.4.1 Enrichment of IBDV WF and ARV s1133 The sup (220 ml) containing propagated IBDV WF or ARV s1133 was added Ultra-15 Amicon tubes (Merck, Germany) and centrifuged at 4,000 × g , 21°C, until all liquid passed through the filter (5–30 min). The tubes were then refilled with additional viral sup and centrifuged again until the entire sup volume passed through. Then, the filters were washed 3 times by adding 15 ml PBS followed by centrifugation. Viruses were then gently re-suspended by pipetting the filters with 15 ml PBS, and transferred to ultracentrifuge tubes (POLYCLEAR, Thermo Fisher Scientific, USA). Viruses were pelleted at 80,000 × g for 2 h, 4°C (F14-6x250 rotor in an ultracentrifuge, Thermo Fisher Scientific, USA). The pellet was re-suspended in 6 ml PBS, filtered through 0.22 µm syringe filters and then stored at -80°C until further use. 2.4.2 Enrichment of aMPV The sup containing propagated aMPV was transferred to ultracentrifuge tubes and pelleted overnight at 38,000 × g , 4°C, using a F14-6x250 rotor in an ultracentrifuge (Thermo Fisher Scientific, USA). The sup was removed, and the pellet was suspended in 18 ml TNE buffer (0.15 M NaCl, 0.025 M Tris, 0.005 M EDTA) + 10% sucrose. Two ultracentrifuge tubes containing 3 phases of different sucrose concentrations dissolved in TNE were prepared; the bottom phase (9 ml) had 65% sucrose, the middle phase (18 ml) had 20% sucrose and the top phase (9 ml) had the viral pellet. The tubes were centrifuged for 6 h at 100,000 ×g, 4°C, in an ultracentrifuge fitted with a F14-6x250 rotor (Thermo Fisher Scientific, USA). Virus pellets were suspended in PBS (5 ml total) and then stored at -80°C until use. 2.4.3 Quantification of viral titer by tissue culture infectious dose 50 (TCID50)/ml assay Viral activity in VERO cells was quantified before and after enrichment. Cells were seeded into 96-cell plates (Greiner Bio-one CELLSTAR, Austria) (2x10 4 cells/well in 100 µl) and incubated overnight. Each plate was infected with one of the tested viruses, in 10-fold dilutions. On day 4 (ARV) or day 8 (aMPV and IBDV WF) after infection, wells were scanned under a light microscope to assess the viral cytopathic effect. TCID50/ml was determined according to the Reed and Muench method [15]. 2.5 Evaluation of viral effect on cancer cells in vitro 2.5.1 Evaluation of cytopathic effect Adherent cell lines were grown in 24-well plates in 0.5 ml medium until > 80% confluence. Medium was replenished and 1:100 (v/v) of a selected virus was added to wells. Cells growing in suspension were maintained in 24-well plates in 1 ml medium, and 1:100 (v/v) of the tested virus was added. Non-infected cells served as controls. The cytopathic effect was evaluated 3–5 days later, under a light microscope. The severity of virus-induced cellular damage was graded as mild when some visible cellular inhibition or scattered cellular damage were noted, medium when an obvious viral cytopathic effect was observed and gaps in the cellular monolayer began to form, or acute when a substantially disrupted monolayer and cellular damage were apparent. 2.5.2 Cell viability assay Cells were seeded in their respective medium, as described above, into 96-well plates (Greiner Bio-one CELLSTAR, Austria) (2x10 3 cells/well in 100 µl) and incubated overnight (37°C and 5% CO 2 ). Each plate was infected with one of the tested viruses, in 10-fold dilutions. Four to five days after infection, 20 µl Cell Titer-Blue reagent (Promega, USA) were added, and plates were further incubated for 4 h. Fluorescence was measured with an Infinite M200 PRO device, 560 nm excitation and 590 nm emission (TECAN, Switzerland). 2.6 Kinetic growth of aMPV in human cancer cell lines MDA-KB2, G361, SW480 and DU145 cancer cell lines and VERO (control cell line) were grown as specified in section ‎2.5.1. Then, aMPV from stock was added 1:100 (v/v). Media samples (10 µl) were collected from cultured cells and stored at 4°C, immediately after virus addition and then after 1, 2, 3, 6, 7, 10 and 14 days. aMPV propagation in the cells was determined by enzyme-linked immunosorbent assay (ELISA), as described below. 2.6.1 Enzyme-linked immunosorbent assay (ELISA) for evaluating aMPV propagation Samples collected from culture medium of aMPV-infected cells were diluted 1:100 (v/v) in coating buffer (0.015 M carbonate/bicarbonate buffer, pH = 9.6), added to 96-well plates (Thermo Fisher Scientific, USA ) and incubated overnight in 4°C. Thereafter, plates were rinsed three times with PBS + 0.05% Tween 20 (PBST) and blocked for 2 h, at 37°C with blocking buffer (5% skim milk in PBST). Afterwards, plates were washed three times with PBST and incubated for 2 h (37°C) with turkey serum raised against aMPV (kindly provided by Phibro Biological Laboratories, Israel). Then, plates were rewashed and incubated for 1 h (37°C) with goat anti-turkey IgG horseradish peroxidase (HRP) (Southern Biotechnology, USA). After additional washes, SIGMAFAST™ O-phenylenediamine dihydrochloride (OPD; Sigma-Aldrich, Israel) was added and absorbance was measured at 450 nm with a Multiskan RC plate reader (Thermo Fisher Scientific, USA). 2.6.2 Titration of aMPV in SW-480 cell line To further evaluate the ability of aMPV to proliferate in SW-40 cells, cells were grown in 24-well plates, as described in Section ‎2.5.1. Then, 50 µl aMPV diluted ten-fold up to 10 6 in cell medium, were added. Plates were incubated (37°C and 5% CO 2 ) for 7 days, after which, the virus was extracted from cells following three freeze-thaw cycles. Samples were analyzed for aMPV propagation by TCID50/ml and ELISA, as described above. 2.7 Animal experiments Animal experiments were conducted with the approval of the Israeli ethics committee (ethic numbers IL-19-5-251 and IL-19-2-98). Animals were maintained under standard light and temperature conditions with ad libitum water and food. To avoid cross-contaminations, each virus treatment group was housed isolated in separate cages. AVs used for animal experiments were propagated, enriched and quantified as described in section 2.4. Virus quantities used are displayed in Fig. 2 A. 2.7.1 AVs treatment of nude mice bearing an induced G361 human melanoma Human melanoma G361 cells were injected subcutaneously (s.c.) (1x10 6 cells/mice) into the right flank of 8-10-week-old female nude mice (Envigo, Israel). On day 10 after tumor induction, upon visualizing pulpable yet too small to be measured tumors, animals were randomly divided into four treatment groups and 100 µl of ARV-s1133, IBDV-WF, aMPV-P20 or PBS as control were intratumorally (i.t.) injected. On day 20, groups receiving viruses were injected with a second 100 µl dose containing a cocktail of all three viruses (1:1:1 v/v from the stock viral solutions following their propagation in VERO cells) and the control group received a second PBS dose. Tumor volumes were measured tri-weekly by caliper. Mice were euthanized upon tumor reaching maximal size in accordance with ethics regulation (12mm diameter). 2.7.2 AVs treatment of immunocompetent BALBc mice bearing an induced 4T1 mammary carcinoma Mouse mammary cancer 4T1 cells were injected s.c. (5x10 4 cells/mice) into the right flank of 8-10-week-old female BALBc mice (Envigo, Israel). On day 5 after tumor induction, upon visualizing pulpable yet too small to be measured tumors, 6 mice per group were injected i.t. with 100 µl of ARV-s1133, IBDV-WF, aMPV-P20 or PBS as control. Treatments were administered again 7 and 14 days later. Blood samples were collected at baseline and 6 days after each treatment. Tumor volumes were measured tri-weekly by caliper. Mice were euthanized upon tumor reaching maximal size in accordance with ethics regulation (12mm diameter). AVs treatment of immunocompetent C57BL6 mice bearing an induced MC38 colon carcinoma Mouse colon cancer MC38 cells were injected s.c. (4x10 5 cells/mice) into the right flank of 8–10 weeks old female C57BL6 mice (Envigo, Israel). On day 7 after tumor induction, upon visualizing pulpable yet too small to be measured tumors, 6 mice per group were injected i.t. with 100 µl of ARV-s1133, IBDV-WF, aMPV-P20 or PBS as control. Treatment was administered again 7 and 14 days later mice. Blood samples were collected at baseline and 6 days after each treatment. Tumor volumes were measured tri-weekly by caliper. Mice were euthanized upon tumor reaching maximal size in accordance with ethics regulation (12mm diameter). 2.7.3 Evaluation of anti-viral humoral immune response by ELISA Blood samples from treated mice were allowed to clot overnight at 4 ºC, after which, sera were separated. To assess the humoral immune responses raised against ARV s1133 and IBDV WF, 96-well ELISA plates (Thermo Fisher Scientific, USA) were coated overnight at 4 ºC with sigma C [16] or VP2 [17] polypeptides, respectively. Then, following washes and blocking (as specified in Section ‎2.6.1), mouse sera diluted 1:100 were added and incubated 1 h at 37 ºC. After additional washes, plates were incubated 1 h at 37 ºC with goat anti-mouse peroxidase (Sigma, Israel), then washed, stained with OPD and absorbance was measured at 450 nm. To test responses raised against aMPV, a commercial ELISA kit (IDEXX Laboratories, USA) was used per manufacturer’s instructions, with the exception of substitution of the kits anti-chicken secondary antibodies with the above-mentioned anti-mouse antibodies. For technical reasons ELISA results for the determination of anti-aMPV antibody levels were not satisfactorily achieved. 2.8 Statistical analysis Statistical significance was assessed using student’s T test, analysis of variance (ANOVA) Tukey or ANOVA Dunnett test, as indicated in figure legends. Statistical analyses were performed using GraphPad Prism software. 3 RESULTS 3.1 Oncolytic activity of avian viruses (AVs) in vitro Eight AVs, from four viral families, were evaluated in vitro for their oncolytic potential. The AVs included two strains of ARV, three strains of IBDV and two strains of IBV and one strain of aMPV. Their effects were tested in the VERO cell line, FF normal human fibroblasts and 18 cancer cell lines from human or mouse origin (Table 1 ). ARV strains s1133 and ISR5215, IBDV-WF and aMPV-P20 displayed effectively infected and damaged VERO cells and some cancer cell lines, but caused no apparent damage to the normal FF culture (Table 1 ). While the attenuated ARV-s1133 vaccine strain caused various levels of damage to 14 out of the 18 cancer cell lines tested, the virulent ARV-ISR5215 field-isolated strain caused milder damage to five cell lines only (Fig. 1 A and Table 1 ). Notably, all viruses used for this experiment were grown to their maximal potential in the VERO cells and used without further quantification. Therefore, viruses can be compared for their effect on distinct cell lines but not with each other. The aMPV-P20 strain affected 9 out of 18 cancer cell lines (Table 1 and as demonstrated in Fig. 1 A and 1 B). Of the three IBDV strains tested, only IBDV-WF mildly affected VERO cells, and induced acute damage to human melanoma cell lines M-15 and M-373, mild effect on G361 and moderate damage to mouse mammary (4T1) and renal (RenCa) cancer cell lines (Table 1 and as demonstrated in Fig. 1 A and 1 C). Both IBV strains tested imparted no visible cytopathic effect on any of the cells used in this study, except for a mild effect of IBV-M41 on LNCaP cells (Table 1 ), and therefore were excluded from further testing. Notably, the two ALL cell lines, REH and 697, seemed entirely resistant to all viruses, while all other cancer cell lines used were affected by at least one of the viruses tested (Table 1 ). Table 1. Cytopathic effect of stock viruses assessed in cell lines Avian reovirus (ARV), Infectious bursal disease virus (IBDV) Infectious bronchitis virus (IBV), Avian Metapneumovirus (aMPV). Acute effect (+++), medium effect (++), mild effect (+), inconclusive, non-repeating effect (±), No apparent effect (-), not tested (nt). * Foreskin fibroblasts are primary culture. 3.2 AV enrichment post-propagation in VERO cells The three AVs inducing the most damage to cancer cell lines (Table 1 ) namely, ARV-s1133, IBDV-WF and aMPV-P20, were propagated in large volumes in VERO cells and then enriched by ultracentrifugation. All three AVs displayed the ability to propagate in VERO cells, and TCID 50 /ml values of 6.4, 5 and 3 for ARV-s1133, IBDV-WF and aMPV-P20, respectively, were reached (Fig. 2 A). TCID 50 /ml values after concentration enrichment were elevated by 5.6, 4.3 and 3.3 logs for ARV-s1133, IBDV-WF and aMPV-P20, respectively (Fig. 2 A). AV activity and its increase post-concentration enrichment were also confirmed by reduction in the viability of VERO cells (Fig. 2 B). Quantified viruses in Fig. 2 A were stored and later used in all mice experiments. 3.3 aMPV-P20 propagation in cancer cells To evaluate the correlation between the virus-induced cytopathic effect and viral ability to propagate and release from cancer cells, aMPV was allowed to infect four cancer cell lines which were acutely (G361 and SW480), mildly (DU145) or not (MDA-KB2) affected by the virus (Table 1 ). Cell culture media samples containing extracellular aMPV-P20 which were released from infected cells were collected over 14 days and viral levels were quantified by ELISA. A significant increase in extracellular aMPV-P20 levels was detected over time in the SW480 culture, and reached levels similar to those measured in the control VERO cells (Fig. 3 A). No increase was evident in MDA-KB2 and DU145 cells, for which a low or no cytopathic effect was observed after infection. Notably, no propagation was detected in the G361 melanoma cells despite the acute cytopathic effect induced by the virus (Fig. 3 A). To further substantiate the ability of aMPV to propagate in the SW480 cell line, cell cultures were treated with ten-fold dilutions of the virus, and incubated for 7 days, after which, virus levels were quantified by ELISA and by TCID50/ml. A significant increase in virus levels and activity was detected both by ELISA and TCID50/ml (Figs. 3 B and 3 C, respectively) in the presence of cells as compared to cell-free samples. TCID50/ml indicated that aMPV-P20 levels increased by more than 7 logs in the SW480 cells. Taken together, these results indicate that aMPV-P20 can propagate in the SW480 cell line while also inflicting a cytopathic effect. 3.4 AV influence on human melanoma cell G361 tumors in nude mice Out of all the AV strains tested in vitro , ARV-s1133, aMPV-P20 and IBDV-WF displayed the widest ability to induce cytopathic and cancer-cell-killing effects (Fig. 1 and Table 1 ) and were therefore chosen for further testing in vivo. First, we evaluated the chosen AVs ability to induce tumor suppression on nude mice bearing a s.c. human melanoma G361 tumors. In a preliminary small scale study, mice were injected i.t. on day 10 after tumor inoculation with a single AV or PBS (Fig. 4 A). Inhibition of tumor growth compared to the PBS control treatment was observed for all three AVs as soon as 7 days after the first treatment. On day 10 after the first treatment, the measured mean tumor volumes (± SD) were 223.7 mm 3 (± 25.9), 169.7 mm 3 (± 36.1) and 196.8 mm 3 (± 80.3) for ARV-s1133, IBDV-WF and aMPV-P20 respectively, as compared to 434.1 mm 3 (± 607.6) for the PBS treatment (Fig. 4 B). These measurements signify a percent increase (± SD) of 264.7% (± 12.9), 229.9% (± 107.9) and 174.3% ± (18.6) for ARV-s1133, IBDV-WF and aMPV-P20 respectively, as compared to 1456.4% (± 1936.7) for the PBS treatment (Fig. 4 C). Of note, although virus treatments displayed substantial and unified inhibition of tumors progression, the effect was not statistically significant due to the high variability in the control group. A second treatment was administered on day 20, with the PBS control group given a second PBS dose while all other groups were injected with a cocktail of all three AVs in order to test the combined viral effect on the tumors (Fig. 4 A). The second dose of AVs significantly inhibited tumor progress in the mice treated with ARV-s1133 and aMPV-P20 as compared to the PBS-treated group while non statistically significant reduction was evident in the IBDV-WF treated group. A 341.5% (± 143.6) increase in tumor size was measured in the PBS treated group 20–21 days (day 41) after administration of the second dose, while tumors in the ARV-s1133-, IBDV-WF- and aMPV-P20-treated groups increased by only 10.2% (± 67.6), 129.9% (± 95.7) and 79.4% (± 77.9), respectively (Fig. 4 D). 3.5 AV influence on murine mammary carcinoma 4T1 cells and tumors in immunocompetent BALBc mice As the Achilles' heel of virotherapy is neutralization by the immune system, the ability of AVs to inhibit tumor growth in immunocompetent mice was also assessed. Mouse 4T1 mammary cells were similarly affected in vitro by all three AVs and exhibited a concentration-dependent reduction in cell viability, reaching approximately 50% at the highest viral concentrations (Fig. 5 A). Notably, although acute (ARV-s1133) and medium (IBDV-WF, aMPV-P20) cytopathic effects were visually demonstrated (Table 1 ), a 10-fold dilution of each of the viruses seemed to diminish their influence on the cells in the cell viability assay (Fig. 5 A). 4T1 cells were s.c. injected into BALBc mice. When tumors were palpable (on day 5) mice were i.t. administered either ARV-s1133, IBDV-WF, aMPV-P20 or PBS, followed by identical doses 7 and 14 days thereafter. A significant increase in the levels of antibodies raised against ARV-s1133 (Fig. 5 B) and IBDV-WF (Fig. 5 C) was detected after the second and third treatment doses, respectively. A significant decrease in tumor progression was seen in the animals treated with ARV-s1133 and IBDV-WF starting 2 days after administration of the second dose (day 14), while in the group treated with aMPV-P20, tumor progression resembled that of the PBS-treated negative control group (Fig. 5 D). 3.6 AV influence on murine colon carcinoma MC38 cells and tumors in immunocompetent C57BL/6 mice. To further establish the potential of AVs, another cancer model was studied in immunocompetent C57BL/6 mice. MC38 cell viability was significantly lowered in vitro when infected with the AVs (Fig. 6 A). ARV-s1133 and aMPV-P20 enrichment significantly increased viral potency as reflected by a reduction in MC38 cell viability, while IBDV-WF displayed similar activity before and after enrichment (Fig. 6 A). MC38 cells were s.c injected into C57BL/6 mice. When tumors were palpable (day 7), ARV-s1133, IBDV-WF, aMPV-P20 or PBS was i.t. injected, and second and third doses were administered 7 and 14 days thereafter. Similar to the observed response in the 4T1-BALBc model, a significant increase in the serum antibodies raised against ARV-s1133 (Fig. 6 B) and IBDV-WF (Fig. 6 C) was detected in the MC38-C57BL6 model after the second and third treatment dose, respectively. Although not significant, a trend of decrease in tumor progression was already evident on the day of the second IBDV-WF and aMPV-P20 treatment dose (Fig. 6 D). Notably, ARV-s1133 which was very potent in inhibiting 4T1 tumor progression in vivo and reducing both 4T1 and MC38 cell viability in vitro , displayed only a mild inhibitory effect on the MC38 tumors. Corresponding to tumor progression, mouse survival was significantly prolonged in the aMPV-P20-treated group as compared to the PBS-treated group. Survival of IBDV-WF-treated mice was also extended, but not significantly (Fig. 6 E). 4 Discussion OVs have gained considerable attention as a potential therapeutic modality for cancer treatment due to their ability to selectively target and destroy cancer cells while sparing normal cells [18]. Various natural or attenuated as well as genetically modified OVs from human or animal origin, including adenoviruses, herpes simplex viruses and reoviruses, have been tested in preclinical and clinical studies [18]. Clinical trials have demonstrated the potential of OVs as cancer therapeutics for the treatment of melanoma [19], breast cancer [20], ovarian cancer [21] and others [22]. In this study, viruses of avian origin were tested to identify those that can selectively destroy cancer cells of mouse and human origin. Out of the eight tested AVs of four different viral families, four viruses induced acute cytopathic effects in one or more cancer cell lines, while normal fibroblasts remained intact. Several mechanisms of action determining susceptibility versus resistance of cells to OVs were described e.g. lack of PKR related RNA degradation due to overactivation of Kras. The differences in susceptibility of several cell lines seen in this study may be explained by presence or lack of such mechanisms relevant to the viruses used in this study. Out of the two ARV strains that induced a cytopathic effect in cancer cells, the attenuated strain s1133 induced a cytopathic effect in 14 of the 18 cancer cell lines tested, while the virulent ISR5215 strain caused milder damage to five lines only. Similarly, the attenuated vaccine IBDV-WF strain was superior to the virulent strain in inducing a cytopathic effect in two human melanoma cell lines and lung and breast cancer mice cell lines. These findings indicate that virus virulence in poultry does not correlate with their effects on cancer cells in mammals. Kim et al. demonstrated that prolonged exposure of human HT1080 fibrosarcoma cells to reovirus yielded high levels of virus-resistant cells [23]. The variety of potential OVs available, that differ in their origin, and possibly possessing different mechanisms of action, and the possibility of employing combinations of viruses as cocktails or in succession, may successfully circumvent development of cancer resistance during treatment. The ARV-s1133, IBDV-WF and aMPV-P20 viruses, which displayed the most pronounced effects on cancer cells, were further evaluated in vitro and in vivo for their potential as OV. Methods for viral propagation and enrichment were developed for each individual AV. Propagation of aMPV in SW480 colon cancer cell line, as established in Fig. 3 A and affirmed in Figs. 3 B and 3 C, was accompanied by an acute virus-induced cytopathic effect. In MDA-Kb2 cells, neither a cytopathic effect nor viral propagation were documented. While viral propagation was expected to contribute to cell destruction, aMPV induced a mild and acute viral cytopathic effect in DU145 and G361 cell lines, respectively, despite their failure to propagate in these cells. This could indicate that other mechanisms for virus-induced cell destruction, such as apoptosis or others are also in play, and may contribute to the anti-cancer viral activities of the AVs. Three mouse models were used to evaluate the oncolytic effects of AVs. The oncolytic effect of the viruses was first evaluated in nude mice carrying G361 human melanoma tumors. The absence of a fully functional acquired immune system in this model enabled assessment of the influence of AVs on human cancers. Differences between the effect of all three AVs on tumors and the PBS negative control were already observed after the first dose. These differences increased and became statistically significant following administration, on day 20 following tumor induction, of the second treatment dose of AVs cocktail in the groups readministered with the aMPV-P20, and the ARV-s1133. Similar tumor inhibition trends were seen for all three AVs treated groups. The contribution of each AV effect on tumor inhibition in this experiment was evaluated only after the first dose. Following administration of the cocktail as a second dose, the effect shown may result from the combined activity of all 3 AVs. The effect of each single AV on tumor inhibition was evaluated and demonstrated in later experiments (Fig. 5 and Fig. 6 ) The ability of viral treatments to retain their effectiveness in parallel to the development of a debilitating targeted immune response was tested in immunocompetent BALBc and C57BL6 mice carrying 4T1 mammary and MC38 colon cancers, respectively. These cells were selected for testing in vivo after their observed susceptibility to AVs in vitro . Antibodies toward ARV-s1133 and IBDV-WF were detected following the second and third intra-tumoral virus doses, respectively. Elevation of antibody levels was timed closely with increased tumor progression rate in AV treated mice and may explain the decreased efficacy of their oncolytic effect. The immune response toward OVs has two faces. On the one hand, the local immune response toward the virus and the infected cancer cells may activate an anti-tumor immune response in the tumor microenvironment [24,25]. This may enhance the efficacy of treatment, since, in addition to oncolysis, the tumor-specific immune response is raised due to presence of viral epitopes and induction of innate and cytotoxic T cell immunity in the tumor microenvironment. This response may add to the destruction of tumor cells, and initiate an immune response against metastasis [26]. On the other hand, after several doses, humoral immune response may neutralize the virus.Pre-existing or newly developed neutralizing antibodies recognizing the virus may disable the treatment over multiple dosing [27,28]. For example, Russell et al. reported that when attempting to use an attenuated measles virus vaccine strain engineered to target CD46 that is abundant on human tumor cells, the major impediment to the treatment efficacy was the pre-existing anti-measles immunity [28]. One way to overcome the immune response is by direct injection of the virus into the tumors [29], which prevents spillover of the vector to other tissues. A possible disadvantage of this approach is the probable limitation of the treatment to the tumor, which may not enable treatment of metastases. Viral migration from the injected tumor site may elicit neutralizing antibodies which will limit treatment effectiveness to the metastasis. A combined treatment of oncolytic virus with immuno-suppressive drugs was suggested to overcome the immune response and to induce metastatic tumor regression [30]. In the current study, three viruses from three different families, were found to induce tumor cell destruction in vitro . In addition to the ARV-s1133 vaccine strain, a field isolate was also tested, and its ability to kill cancer cells in vitro was demonstrated. The genetic variation of ARV is determined by the sequence of sigma C. This protein induces the production of neutralizing antibodies, and its variation enables the virus to escape the immune response [31]. The high variability of ARV generates different strains that evade neutralizing antibodies raised against other strains [32,33], which ultimately highly enriches the arsenal of viruses that can be used for treatment. Induction of an immune response against the tumor while enabling continuation of the treatment by switching the treatment viruses, allows for combined and prolonged tumor attack both by the virus and the immune system. The growth of 4T1 tumors was significantly inhibited following treatment with ARV-s1133 and IBDV-WF. The growth of colon cancer cell line MC38 tumors was significantly inhibited and survival of mice bearing s.c. MC38 tumors was extended following treatment with aMPV-P20 and IBDV-WF. Following repeated AV administrations, tumors began to re-grow in correlation with specific anti-virus antibody titers. A possible route to overcome decrease of treatment efficacy due to the rise of the neutralizing immune response may be to simultaneously or sequentially administer other OVs. No clinical adverse effects, including body weight loss or abnormal appearance and behavior, were observed as result of virus administration in any of the tested mouse models (data not shown). These viruses are non-zoonotic and are widely used as commercial live attenuated vaccines in poultry. No evidence of adverse risk for farmers who are constantly exposed to those viruses was reported. In conclusion, this study characterized the oncolytic potential of three AVs. The AVs induced cytopathic effects and effectively inhibited cancer cell line proliferation in vitro and delayed tumor growth in vivo . The AVs along with their immunogenetically distinct field variants, bear potential to serve as a vast arsenal of OVs to enable prolonged and effective treatment of susceptible cancers. Declarations Ethics statement Animal experiments were conducted with the approval of the Israeli ethics committee (ethic numbers IL-19-5-251 and IL-19-2-98). Funding: This research was funded internally by MIGAL institute. ACKNOWLEDGMENT. The authors would like to thank Prof. Jamal Mahajna from MIGAL institute in the Galilee for his support, and Prof. Michal Lotem from Hadassah Medical Center in Jerusalem for melanoma cells. References Jin, K.-T.; Du, W.-L.; Liu, Y.-Y.; Lan, H.-R.; Si, J.-X.; Mou, X.-Z.; Marchini, A.; Ilkow, C.S.; Melcher, A. Cancers Oncolytic Virotherapy in Solid Tumors: The Challenges and Achievements Oncolytic Virotherapy in Solid. 2021 , doi:10.3390/cancers13040588. Chaurasiya, S.; Fong, Y.; Warner, S.G.; Bartlett, D.L. Oncolytic Virotherapy for Cancer: Clinical Experience. Biomedicines 2021, 9 . Yang, L.; Gu, X.; Yu, J.; Ge, S.; Fan, X. Oncolytic Virotherapy: From Bench to Bedside., doi:10.3389/fcell.2021.790150. Abd-Aziz, N.; Poh, C.L. Development of Oncolytic Viruses for Cancer Therapy. Transl. Res. 2021 , 237 , 98–123, doi:10.1016/J.TRSL.2021.04.008. Chaurasiya, S.; Fong, Y.; Warner, S.G. Oncolytic Virotherapy for Cancer: Clinical Experience. Biomedicines 2021, 9 . Cesaro, T.; Michiels, T. 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Oncolytic Virus Immunotherapies in Ovarian Cancer: Moving beyond Adenoviruses. Porto Biomed. J. 2018 , 3 , e7, doi:10.1016/J.PBJ.0000000000000007. Macedo, N.; Miller, D.M.; Haq, R.; Kaufman, H.L. Clinical Landscape of Oncolytic Virus Research in 2020. J. Immunother. Cancer 2020 , 8 , 1486, doi:10.1136/JITC-2020-001486. Kim, M.; Egan, C.; Alain, T.; Urbanski, S.J.; Lee, P.W.; Forsyth, P.A.; Johnston, R.N. Acquired Resistance to Reoviral Oncolysis in Ras-Transformed Fibrosarcoma Cells. Oncogene 2007 , 26 , 4124–4134, doi:10.1038/SJ.ONC.1210189. Worschech, A.; Haddad, D.; Stroncek, D.F.; Wang, E.; Marincola, F.M.; Szalay, A.A. The Immunologic Aspects of Poxvirus Oncolytic Therapy. Cancer Immunol. Immunother. 2009 , 58 , 1355–1362, doi:10.1007/S00262-009-0686-7. Toda, M.; Rabkin, S.D.; Kojima, H.; Martuza, R.L. Herpes Simplex Virus as an in Situ Cancer Vaccine for the Induction of Specific Anti-Tumor Immunity. Hum. Gene Ther. 1999 , 10 , 385–393, doi:10.1089/10430349950018832. 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Cite Share Download PDF Status: Published Journal Publication published 21 Feb, 2026 Read the published version in Archives of Virology → Version 1 posted Editorial decision: Major Revision 09 Aug, 2025 Reviewers agreed at journal 04 Jun, 2025 Reviewers invited by journal 27 May, 2025 Editor assigned by journal 21 May, 2025 First submitted to journal 18 May, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6690670","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":462699717,"identity":"fd36c3a9-e9ae-4257-a6b9-44ebcdb99dd4","order_by":0,"name":"Jacob Pitcovski Pitcovski","email":"","orcid":"","institution":"Migal Galilee Technology Center","correspondingAuthor":false,"prefix":"","firstName":"Jacob","middleName":"Pitcovski","lastName":"Pitcovski","suffix":""},{"id":462699718,"identity":"7b98db0f-5368-4fcc-9ca9-88093fe06857","order_by":1,"name":"Gilad Gallili","email":"","orcid":"","institution":"Phibro Israel","correspondingAuthor":false,"prefix":"","firstName":"Gilad","middleName":"","lastName":"Gallili","suffix":""},{"id":462699719,"identity":"6f20b147-03a3-443c-9e79-4fd3a7447b94","order_by":2,"name":"Daria Oren Aharon","email":"","orcid":"","institution":"Tel-Hai Academic College: Tel Hai Academic College","correspondingAuthor":false,"prefix":"","firstName":"Daria","middleName":"Oren","lastName":"Aharon","suffix":""},{"id":462699720,"identity":"59a59d47-ec83-461f-b7fb-fae5b24f8987","order_by":3,"name":"Shir Malka","email":"","orcid":"","institution":"Tel-Hai Academic College: Tel Hai Academic College","correspondingAuthor":false,"prefix":"","firstName":"Shir","middleName":"","lastName":"Malka","suffix":""},{"id":462699721,"identity":"012a5a61-38a5-4fc8-884c-51b9e7f7c8b8","order_by":4,"name":"Gal Yanovich","email":"","orcid":"","institution":"Tel-Hai Academic College: Tel Hai Academic College","correspondingAuthor":false,"prefix":"","firstName":"Gal","middleName":"","lastName":"Yanovich","suffix":""},{"id":462699722,"identity":"b4f31d4a-4ba3-4375-8fd7-259d63c20775","order_by":5,"name":"Elad Milrot","email":"","orcid":"","institution":"Migal Galilee Technology Center","correspondingAuthor":false,"prefix":"","firstName":"Elad","middleName":"","lastName":"Milrot","suffix":""},{"id":462699723,"identity":"2f9f9c14-867f-4084-88c7-dbe87b5a7e63","order_by":6,"name":"Ehud Shahar","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA50lEQVRIiWNgGAWjYDACCSBibGCQY2AGch6AhRgbDxCjxRisJQGipYEoLYkNDHAtDAx4tfBL9xje/LnDLn1tO/vFDwkMNvnyDsz4bZGcc8bYmvdMcu62wzzFEgkMaZYbDxBwmMGNHDNpxjZmkJYEoJbDBoYNBLTYA7VI/myrTzc7zJP8gygtBhI5ZhK8bYcTzA6zHwPbIk8oxCRupBVb87YdNwQ6jM0iwSDNwICZgBb+Gckbb/5sq5Y3O3/88Y0PFTYG8u3tDx/g04IEeAyA7gSiw0SqBwJ2iNnyDcRrGQWjYBSMgpEBAAasTDGTm58+AAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-8397-1889","institution":"Migal Galilee Technology Center","correspondingAuthor":true,"prefix":"","firstName":"Ehud","middleName":"","lastName":"Shahar","suffix":""}],"badges":[],"createdAt":"2025-05-18 08:51:40","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6690670/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6690670/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00705-026-06559-8","type":"published","date":"2026-02-21T15:58:53+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":83860960,"identity":"4e4709ce-a88f-4d90-b506-b7157e256da1","added_by":"auto","created_at":"2025-06-03 19:21:02","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":252759,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCytopathic effect of avian viruses on cancer cell lines.\u003c/strong\u003e Representative microscopy images, following introduction of viruses to cells at 80% confluence, showing: \u003cstrong\u003eA.\u003c/strong\u003e G361 human melanoma cell line following incubation with (left to right) PBS negative control, ARV s1133 (acute effect), ARV strain ISR5215 (acute effect), aMPV-P20 (acute effect) or IBDV-WF (mild effect). White arrows indicate cytopathic effect of IBDV-WF visible as cellular clumping. \u003cstrong\u003eB.\u003c/strong\u003eLNCaP human prostate carcinoma cell line after incubation with (left to right) PBS negative control, ARV s1133 (acute effect) or aMPV-P20 (acute effect). \u003cstrong\u003eC.\u003c/strong\u003eM-15 human melanoma cell line after incubation with (left to right) PBS negative control or IBDV-WF (acute effect). Magnification X100.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6690670/v1/ade0cdd77c338f67b713e78b.jpg"},{"id":83860955,"identity":"b9b1cfe5-9515-44dd-87e4-f2aa58bb638d","added_by":"auto","created_at":"2025-06-03 19:21:02","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":95020,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eActivity of avian viruses (AV) after propagation in VERO cells and concentration enrichment.\u003c/strong\u003e Samples containing AV were taken from medium after AV propagation in VERO cells, and after AV concentration enrichment by ultracentrifugation. AV activity was assessed by determining \u003cstrong\u003e(A)\u003c/strong\u003e TCID50/ml in VERO cells (2x10\u003csup\u003e4\u003c/sup\u003e cells/well in 100 µl) and \u003cstrong\u003e(B) \u003c/strong\u003ecell viability in VERO cells (2x10\u003csup\u003e3\u003c/sup\u003e cells/well in 100 µl). \u003cstrong\u003eA.\u003c/strong\u003e Bar graph displaying calculated TCID50/ml for samples collected after (left to right) ARV-s1133, IBDV-WF and aMPV-P20 propagation in VERO cells (green) and after enrichment by ultracentrifuge (blue). \u003cstrong\u003eB.\u003c/strong\u003e Graphs displaying cell viability (mean ± SD) of serially diluted samples taken after (top to bottom) ARV-s1133, IBDV-WF and aMPV-P20 propagation in VERO cells (pre-enrichment; green) and post-enrichment (blue) of AVs by ultracentrifugation. Dunnett's ANOVA was used to test statistical significance between uninfected VERO cells versus VERO cells incubated with pre-enrichment samples containing AVs (green asterisks), and uninfected VERO cells versus VERO cells incubated with post-enrichment samples containing AVs (blue asterisks). Paired T test (1 tailed) was used to test statistical significance between VERO cells incubated with samples pre versus post-enrichment (orange asterisks). * p\u0026lt;0.05, ** p\u0026lt;0.01, *** p\u0026lt;0.001, **** p\u0026lt;0.0001\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6690670/v1/674ea70dadd015b6b203aeec.jpg"},{"id":83861268,"identity":"43a92f90-4cdf-4206-a333-4727ca021166","added_by":"auto","created_at":"2025-06-03 19:29:02","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":90866,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eaMPV-P20 propagation in cancer cell lines.\u003c/strong\u003e \u003cstrong\u003eA.\u003c/strong\u003e aMPV-P20 was added to VERO cells, and four cultures of cancer cell lines (as indicated), and its propagation was quantified by ELISA. Graph presents absorption (mean ± SE) at 450 nm of medium collected from cultures immediately after addition of the virus (day 0) and on days 1, 2, 3, 6, 7, 10 and 14 after virus inoculation. aMPV-P20 was detected using turkey serum raised against aMPV and goat anti-turkey IgG-HRP. Asterisks indicate the significance of virus levels in cell culture as compared to the corresponding cell sample collected on day 0. \u003cstrong\u003eB-C. \u003c/strong\u003e\u0026nbsp;aMPV-P20 was added to SW480 cell cultures or SW480 culture medium without cells and incubated for 7 days. Thereafter, plates were frozen and thawed three times and 10 μl samples from each well were collected and serially diluted 10-fold. \u003cstrong\u003eB.\u003c/strong\u003e ELISA was performed as described above, to detect aMPV-P20 in the diluted samples. Graph presents absorption (mean ± SD) at 450 nm. Asterisks indicate the significance of virus levels in samples collected from aMPV-20-infected SW480 cells versus cell-free samples. \u003cstrong\u003eC.\u003c/strong\u003e Graph presents TCID50/ml (mean ± SD) for aMPV-P20 activity in VERO cells. Asterisks indicate the significance of TCID50/ml in samples collected from aMPV-P20-infected SW480 cells versus cell-free samples.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6690670/v1/f67becb2d900da11948b606f.jpg"},{"id":83860956,"identity":"3fbfb1ef-f0ff-46ad-b6ea-6be37da2ccd1","added_by":"auto","created_at":"2025-06-03 19:21:02","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":113264,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIntratumoral AV treatment of nude mice bearing G361 human melanoma tumors.\u003c/strong\u003e \u003cstrong\u003eA.\u003c/strong\u003e Experiment design and time scale. Female nude mice were subcutaneously inoculated on day 0 with G361 human melanoma cells (1x10\u003csup\u003e6\u003c/sup\u003e cells/mouse), and then treated on day 10 with an intra-tumoral injection of either ARV-s1133 (n=3), IBDV-WF (n=3), aMPV-P20 (n=4) or PBS (n=3). On day 20, a second intra-tumoral treatment of either PBS to the control group, or a cocktail of all three AVs was administered to the mice. \u003cstrong\u003eB.\u003c/strong\u003e Graph presenting tumor volume. \u003cstrong\u003eC.\u003c/strong\u003e Graph presenting calculated percent tumor volume increase as compared to its volume on day 1 of treatment (Day 10). \u003cstrong\u003eD.\u003c/strong\u003e Graph presenting percent tumor volume increase as compared to its volume on the day of the second treatment (Day 20). Black arrows indicate administration of treatments. Red asterisks indicate significance of volume increase between PBS control and the ARV-s1133 treatment groups. Blue asterisks indicate significance in tumor volume change between PBS control and the aMPV-P20 treatment groups.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6690670/v1/e074b5c0ad4bd946b163dd35.jpg"},{"id":83860966,"identity":"b961e57a-e7f5-437e-89c7-d4f511fc5d20","added_by":"auto","created_at":"2025-06-03 19:21:02","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":139100,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAV influence on 4T1 mammary carcinoma cells \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and on subcutaneous tumors \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e \u003cstrong\u003eA.\u003c/strong\u003e Graph presenting \u003cem\u003ein vitro\u003c/em\u003e viability (mean ± SD) of 4T1 cells (2x10\u003csup\u003e3\u003c/sup\u003e cells/well in 100 µl) following infection with AVs. 4T1 cells were seeded into 96-well plates and infected with 10-fold dilutions of ARV-s1133 (left), IBDV-WF (middle) or aMPV-P20 (right), and incubated for five days, after which, the Cell Titer-Blue cell viability assay was performed. Top and bottom dashed red lines indicate fluorescence detected in uninfected or no-cell controls, respectively. Asterisks indicate significant reduction in cell viability as compared to the uninfected cells.\u003cstrong\u003e B-D. \u003c/strong\u003eFemale BALBc mice were subcutaneously inoculated on day 0 with 4T1 mouse mammary carcinoma cells (5x10\u003csup\u003e4\u003c/sup\u003e cells/mouse). Starting from day 5, mice received three intratumoral injections at 7-day intervals (indicated by black arrows), of either ARV-s1133 (n=8), IBDV-WF (n=8), aMPV-P20 (n=8) or PBS (n=8).\u0026nbsp; \u003cstrong\u003eB-C. \u003c/strong\u003e\u0026nbsp;Serum collected on days 0, 11, 18 and 25 from treated mice was evaluated by ELISA for the levels of antibodies raised against the (B) ARV-s1133 σC polypeptide or the (C) IBDV-WF VP2 polypeptide. ELISA plates were coated with polypeptide antigens and blocked, before mouse serum was added and later detected with goat anti-mouse-peroxidase. Statistical analysis was performed using ANOVA Dunnett's to determine significance versus the PBS control group.\u003cstrong\u003e D. \u003c/strong\u003eGraphs showing comparison of tumor volume between the PBS-treated negative control and the groups treated with ARV-s1133 (left), IBDV-WF (middle) or aMPV-P20 (right).\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6690670/v1/a383379b680206d98eecaac1.jpg"},{"id":83861269,"identity":"ac4d0824-db92-4a35-ae33-85f6de4d6399","added_by":"auto","created_at":"2025-06-03 19:29:02","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":164624,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAV influence on MC38 colon carcinoma cells \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and on subcutaneous tumors \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e \u003cstrong\u003eA.\u003c/strong\u003e Graphs displaying mean (± SD) MC38 cell (2x10\u003csup\u003e3\u003c/sup\u003e cells/well in 100 µl) viability after infection with 10-fold serially diluted samples of ARV-s1133 (left), IBDV-WF (middle) or aMPV-P20 (right) harvested directly after propagation in VERO cells (pre-enrichment - green) or after enrichment by ultracentrifugation (post enrichment - blue). Asterisks signify a statistically significant difference in viability of cells incubated with pre-enrichment (green) or post-enrichment (blue) AVs samples versus uninfected cells. Orange asterisks signify a statistically significant difference in cell viability between cells incubated with pre-enrichment vs. post-enrichment AVs samples. \u003cstrong\u003eB-E. \u003c/strong\u003eFemale C57BL/6 mice were subcutaneously inoculated on day 0 with MC38 mouse colon carcinoma cells (4x10\u003csup\u003e5\u003c/sup\u003e cells/mouse), and from day 7, received three intra-tumoral injections, at 7-day intervals (indicated by black arrows), with either ARV-s1133 (n=6), IBDV-WF (n=6), aMPV-P20 (n=6) or PBS (n=6).\u0026nbsp; \u003cstrong\u003eB-C. \u003c/strong\u003e\u0026nbsp;Graphs displaying (B) anti-ARV-s1133 σC polypeptide and (C) and anti-IBDV-WF VP2 polypeptide antibody levels measured by ELISA in serum of mice treated with AVs. Serum was collected from treated mice on days 6, 13, 20 and 27. ELISA plates were coated with polypeptide antigens and blocked, then mouse serum was added and later detected with goat anti-mouse-peroxidase. ANOVA Dunnett's was performed to determine significance versus the PBS control group. \u003cstrong\u003eD-E. \u003c/strong\u003eGraphs showing (D) tumor volume and (E) percent survival of mice bearing tumors treated with PBS, ARV-s1133, IBDV-WF or aMPV-P20.\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6690670/v1/ec8a6714dff1743fc6e1aa11.jpg"},{"id":103252713,"identity":"c4498289-49e6-425e-a078-54c474c1d2c5","added_by":"auto","created_at":"2026-02-23 16:15:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2192040,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6690670/v1/69a9d15d-3445-4127-9b7f-73bb694f9fe4.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eViruses of Avian Origin for Cancer Virotherapy\u003c/p\u003e","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eAn oncolytic virus (OV) is defined by its ability to selectively propagate within and suppress tumor cells without affecting normal cells [1]. Several OVs have been tested in clinical studies and appear to be safe at feasible doses [2]. Clinical studies have demonstrated that OVs may significantly improve the prognosis of cancer patients [3]. However, oncolytic virotherapy is still limited by, for example, neutralizing antibodies raised against the therapeutic OV [4]. This phenomenon, as well as others, necessitates new routes for identification and manufacture of OVs for virotherapy.\u003c/p\u003e \u003cp\u003eThe mechanisms by which OV differentiate between normal and cancerous cells is known for some of the viruses, while others are not yet resolved. One well characterized mechanism of RNA-virus selectivity for cancer cells is over-activation of the Ras oncogene leading to under-phosphorylation and subsequent inactivation of the RNA-activated protein kinase (PKR) [5]. In normal cells, PKR acts as both a sensor and an effector in the response to viral infections [6]. After sensing double-stranded RNA molecules in the cytoplasm of infected cells, phosphorylated PKR inhibits mRNA translation, triggers apoptosis, and amplifies the IFN response, resulting in inhibition of virus replication [6].This mechanism of selectivity presents an appealing opportunity for use of RNA viruses which are non-pathogenic or infectious in humans as therapeutic OVs.\u003c/p\u003e \u003cp\u003eOV can be classified as genetically engineered (e.g., Talimogene laherperepvec (T-VEC)) or natural (e.g., reovirus) viruses. Reovirus type 3 isolated from humans induces direct lysis of cancer cells, as well as antitumor immune activation of dendritic cells, natural killer (NK) cells, and effector T-cells [7]. On this basis, a human reovirus treatment, Reolysin, was developed and tested in multiple clinical trials [5] as monotherapy or in combination with other drugs to treat several cancers, including metastatic breast cancer, advanced-stage head and neck cancer, metastatic ovarian cancer, malignant gliomas, prostate cancer and metastatic melanoma [5,8]. Another natural OV is the Newcastle disease virus (NDV), an avian pathogen which is a member of the paramyxovirus family. The oncolytic potential of both virulent and attenuated NDV strains has been demonstrated in cell cultures, in experimental animal models, and in clinical trials. Preclinical and clinical experience with oncolytic NDV has indicated its potential efficacy for treatment of a variety of lymphomas and solid tumors, including metastases [9]. Freeman et al. reported on the potential efficacy of NDV as a treatment for glioblastoma [10]. As with reovirus, both a direct cytopathic effect and an indirect effect through induction of the immune system were demonstrated for NDV [9].\u003c/p\u003e \u003cp\u003eThe current work aimed to evaluate avian metapneumoviruses (aMPV), avian reovirus (ARV), infectious bursal disease virus (IBDV) and infectious bronchitis virus (IBV) for their ability to serve as OVs. The four avian RNA viruses are pathogenic in poultry, but with no documented pathogenicity in humans. aMPV, a member of the Paramyxoviridae family, is an enveloped virus that carries a single-stranded, negative-sense RNA genome [11]. ARV, a member of the Reoviridae family, is a non-enveloped virus with 10 segments of a double-stranded RNA (dsRNA) genome [12]. ARV variants may be divided into four immunogenically distinct genotype clusters which can further extend the OVs arsenal [13]. IBDV, a member of the Birnaviridae family, is a nonenveloped virus that carries two dsRNA segments [14,15]. IBV, a member of the Coronaviridae family, is an enveloped positive-strand RNA virus [11].\u003c/p\u003e"},{"header":"2 MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Cells\u003c/h2\u003e \u003cp\u003eThe cell lines VERO (Green monkey epithelial kidney), SK-BR-3, MCF-7, MDA-KB2, MDA-MB231(human breast cancer), G361 (human melanoma), DU145, PC3 (human prostate cancer), SW480 (human colon cancer), B16-F10 (mouse melanoma), 4T1 (mouse mammary cancer), LLC1 (mouse Lewis lung cancer) and RENCA (mouse renal cancer) were obtained from the American Type Culture Collection (ATCC, USA). Cell lines 697, REH (acute lymphoblastic leukemia [ALL]) and LNCAP (human prostate cancer) were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Germany). Human foreskin fibroblasts primary tissue culture (FF) was kindly provided by Prof. Raphael Gorodetsky of the Sharett Institute Jerusalem. Melanoma lines M-15, M-373, M-374 and M-376 were a kind gift from Prof. Michal Lotem at the Sharett Institute of Oncology, Hadassah Medical Center, Jerusalem. MC-38 (mouse colon cancer) was obtained from the Technion, Israel.\u003c/p\u003e \u003cp\u003eFF cells were grown in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM) 4.5 g/l glucose supplemented with 10% fetal calf serum (FCS), 1% L-glutamate, 1% sodium pyruvate 1% MEM non-essential amino acids, 1% MEM vitamin solution and 1% penicillin-streptomycin solution (Biological Industries, Israel). M-15, M-373, M-374 and M-376 cells were grown in DMEM\u0026thinsp;+\u0026thinsp;RPMI (1:1) supplemented with 10% FCS, 1% L-glutamate, 1% sodium pyruvate and 1% penicillin-streptomycin solution. MC-38 cells were grown in DMEM supplemented with 10% FCS, 1% L-glutamate, 1% sodium pyruvate, 1% MEM non-essential amino acids, 1% HEPES and 1% penicillin-streptomycin solution. All other cell lines were maintained under conditions and in the media recommended by ATCC or DSMZ. Cells were incubated at 37\u0026deg;C and in 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Viruses tested\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1 Avian reoviruses (ARV)\u003c/h2\u003e \u003cp\u003eReovirus field isolate 5215 was collected from the tendon of infected birds. The tendon fluid was used to infect (100 \u0026micro;l) specific-pathogen-free embryos, and the virus was isolated by mixing 1:1 (v/v) of the allantoic fluid with lysis buffer (10 mM Tris pH 8.0, 10 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 100 mM NaCl), freeze-thawing three times, and centrifuging (10,000 \u0026times;\u003cem\u003eg\u003c/em\u003e, 4\u0026deg;C). Viral presence was determined as described previously by Goldenberg et al. [32]. Briefly, RNA was extracted and concentrated from the supernatant then reverse transcribed and sequenced. The supernatant was stored at 4\u0026deg;C until use. Reovirus s1133, an attenuated vaccine strain, was supplied by Phibro Vaccines (Israel) and propagated in VERO cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2 Infectious bursal disease virus (IBDV)\u003c/h2\u003e \u003cp\u003eIBDV KS is an Israeli-isolated pathogenic strain, previously characterized and sequenced by Pitcovski et al [12]. IBDV MB is an attenuated vaccine strain, derived from the KS strain [13] and manufactured by Phibro Vaccines (Israel). IBDV Winterfield (IBDV WF; Solvay, Belgium) is an attenuated mild vaccine strain [14], and was propagated in VERO cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.2.3 Avian metapneumovirus (aMVP)\u003c/h2\u003e \u003cp\u003eaMPV P20 is an attenuated vaccine strain (supplied by Phibro Vaccines, Israel) and was propagated in VERO cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.2.4 Infectious bronchitis virus (IBV)\u003c/h2\u003e \u003cp\u003eIBV M41 a virulent strain and IBV H120 is a live attenuated vaccine derived from strain M41 (supplied by Phibro Vaccines, Israel). Both were propagated in embryonated eggs and quantified by the supplier.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.2.5 Preparation of virus stock banks\u003c/h2\u003e \u003cp\u003eViruses received lyophilized were suspended according to the manufacturer\u0026rsquo;s instructions. All liquid virus solutions were aliquoted and stored at -80\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Virus propagation in VERO cells\u003c/h2\u003e \u003cp\u003eVERO cells were grown to 90\u0026ndash;100% confluence in 175 cc\u003csup\u003e2\u003c/sup\u003e flasks. Growth medium was removed, and adherent cells were added with 5 ml of either ARV s1133, IBDV WF or aMPV-P20. Before adding to the cells, the ARV s1133, IBDV WF or aMPV-P20 [at tissue culture infectious dose 50 (TCID\u003csub\u003e50\u003c/sub\u003e)/ml of 8, 7.32 and 5, respectively] were diluted 5 folds in cell infection medium (growth medium with FCS reduced to 0.5%). After a 1-hour incubation, 39 ml cell infection medium were added to flasks and cells were further incubated. On days 4 (ARV) or 8 (IBDV and aMPV), the cytopathic effect was verified under the microscope. Flasks were frozen (-80\u0026deg;C) and thawed (37\u0026deg;C) twice, after which, all content was collected and centrifuged (9800 \u0026times;\u003cem\u003eg\u003c/em\u003e, 10 min, 4\u0026deg;C for ARV s1133 and IBDV WF or 4000 \u0026times;\u003cem\u003eg\u003c/em\u003e, 15 min, 4\u0026deg;C for aMPV). The supernatant (sup) was collected and stored at -80\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Enrichment and concentration of propagated viruses\u003c/h2\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1 Enrichment of IBDV WF and ARV s1133\u003c/h2\u003e \u003cp\u003eThe sup (220 ml) containing propagated IBDV WF or ARV s1133 was added Ultra-15 Amicon tubes (Merck, Germany) and centrifuged at 4,000 \u0026times;\u003cem\u003eg\u003c/em\u003e, 21\u0026deg;C, until all liquid passed through the filter (5\u0026ndash;30 min). The tubes were then refilled with additional viral sup and centrifuged again until the entire sup volume passed through. Then, the filters were washed 3 times by adding 15 ml PBS followed by centrifugation. Viruses were then gently re-suspended by pipetting the filters with 15 ml PBS, and transferred to ultracentrifuge tubes (POLYCLEAR, Thermo Fisher Scientific, USA). Viruses were pelleted at 80,000 \u0026times;\u003cem\u003eg\u003c/em\u003e for 2 h, 4\u0026deg;C (F14-6x250 rotor in an ultracentrifuge, Thermo Fisher Scientific, USA). The pellet was re-suspended in 6 ml PBS, filtered through 0.22 \u0026micro;m syringe filters and then stored at -80\u0026deg;C until further use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2 Enrichment of aMPV\u003c/h2\u003e \u003cp\u003eThe sup containing propagated aMPV was transferred to ultracentrifuge tubes and pelleted overnight at 38,000 \u0026times;\u003cem\u003eg\u003c/em\u003e, 4\u0026deg;C, using a F14-6x250 rotor in an ultracentrifuge (Thermo Fisher Scientific, USA). The sup was removed, and the pellet was suspended in 18 ml TNE buffer (0.15 M NaCl, 0.025 M Tris, 0.005 M EDTA)\u0026thinsp;+\u0026thinsp;10% sucrose. Two ultracentrifuge tubes containing 3 phases of different sucrose concentrations dissolved in TNE were prepared; the bottom phase (9 ml) had 65% sucrose, the middle phase (18 ml) had 20% sucrose and the top phase (9 ml) had the viral pellet. The tubes were centrifuged for 6 h at 100,000 \u0026times;g, 4\u0026deg;C, in an ultracentrifuge fitted with a F14-6x250 rotor (Thermo Fisher Scientific, USA). Virus pellets were suspended in PBS (5 ml total) and then stored at -80\u0026deg;C until use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.4.3 Quantification of viral titer by tissue culture infectious dose 50 (TCID50)/ml assay\u003c/h2\u003e \u003cp\u003eViral activity in VERO cells was quantified before and after enrichment. Cells were seeded into 96-cell plates (Greiner Bio-one CELLSTAR, Austria) (2x10\u003csup\u003e4\u003c/sup\u003e cells/well in 100 \u0026micro;l) and incubated overnight. Each plate was infected with one of the tested viruses, in 10-fold dilutions. On day 4 (ARV) or day 8 (aMPV and IBDV WF) after infection, wells were scanned under a light microscope to assess the viral cytopathic effect. TCID50/ml was determined according to the Reed and Muench method [15].\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Evaluation of viral effect on cancer cells in vitro\u003c/h2\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e2.5.1 Evaluation of cytopathic effect\u003c/h2\u003e \u003cp\u003eAdherent cell lines were grown in 24-well plates in 0.5 ml medium until \u0026gt;\u0026thinsp;80% confluence. Medium was replenished and 1:100 (v/v) of a selected virus was added to wells. Cells growing in suspension were maintained in 24-well plates in 1 ml medium, and 1:100 (v/v) of the tested virus was added. Non-infected cells served as controls.\u003c/p\u003e \u003cp\u003eThe cytopathic effect was evaluated 3\u0026ndash;5 days later, under a light microscope. The severity of virus-induced cellular damage was graded as mild when some visible cellular inhibition or scattered cellular damage were noted, medium when an obvious viral cytopathic effect was observed and gaps in the cellular monolayer began to form, or acute when a substantially disrupted monolayer and cellular damage were apparent.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e2.5.2 Cell viability assay\u003c/h2\u003e \u003cp\u003eCells were seeded in their respective medium, as described above, into 96-well plates (Greiner Bio-one CELLSTAR, Austria) (2x10\u003csup\u003e3\u003c/sup\u003e cells/well in 100 \u0026micro;l) and incubated overnight (37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e). Each plate was infected with one of the tested viruses, in 10-fold dilutions. Four to five days after infection, 20 \u0026micro;l Cell Titer-Blue reagent (Promega, USA) were added, and plates were further incubated for 4 h. Fluorescence was measured with an Infinite M200 PRO device, 560 nm excitation and 590 nm emission (TECAN, Switzerland).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Kinetic growth of aMPV in human cancer cell lines\u003c/h2\u003e \u003cp\u003eMDA-KB2, G361, SW480 and DU145 cancer cell lines and VERO (control cell line) were grown as specified in section \u0026lrm;2.5.1. Then, aMPV from stock was added 1:100 (v/v). Media samples (10 \u0026micro;l) were collected from cultured cells and stored at 4\u0026deg;C, immediately after virus addition and then after 1, 2, 3, 6, 7, 10 and 14 days. aMPV propagation in the cells was determined by enzyme-linked immunosorbent assay (ELISA), as described below.\u003c/p\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003e2.6.1 Enzyme-linked immunosorbent assay (ELISA) for evaluating aMPV propagation\u003c/h2\u003e \u003cp\u003eSamples collected from culture medium of aMPV-infected cells were diluted 1:100 (v/v) in coating buffer (0.015 M carbonate/bicarbonate buffer, pH\u0026thinsp;=\u0026thinsp;9.6), added to 96-well plates (Thermo Fisher Scientific, USA\u003cem\u003e)\u003c/em\u003e and incubated overnight in 4\u0026deg;C. Thereafter, plates were rinsed three times with PBS\u0026thinsp;+\u0026thinsp;0.05% Tween 20 (PBST) and blocked for 2 h, at 37\u0026deg;C with blocking buffer (5% skim milk in PBST). Afterwards, plates were washed three times with PBST and incubated for 2 h (37\u0026deg;C) with turkey serum raised against aMPV (kindly provided by Phibro Biological Laboratories, Israel). Then, plates were rewashed and incubated for 1 h (37\u0026deg;C) with goat anti-turkey IgG horseradish peroxidase (HRP) (Southern Biotechnology, USA). After additional washes, SIGMAFAST\u0026trade; O-phenylenediamine dihydrochloride (OPD; Sigma-Aldrich, Israel) was added and absorbance was measured at 450 nm with a Multiskan RC plate reader (Thermo Fisher Scientific, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003e2.6.2 Titration of aMPV in SW-480 cell line\u003c/h2\u003e \u003cp\u003eTo further evaluate the ability of aMPV to proliferate in SW-40 cells, cells were grown in 24-well plates, as described in Section \u0026lrm;2.5.1. Then, 50 \u0026micro;l aMPV diluted ten-fold up to 10\u003csup\u003e6\u003c/sup\u003e in cell medium, were added. Plates were incubated (37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e) for 7 days, after which, the virus was extracted from cells following three freeze-thaw cycles. Samples were analyzed for aMPV propagation by TCID50/ml and ELISA, as described above.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Animal experiments\u003c/h2\u003e \u003cp\u003e Animal experiments were conducted with the approval of the Israeli ethics committee (ethic numbers IL-19-5-251 and IL-19-2-98). Animals were maintained under standard light and temperature conditions with ad libitum water and food. To avoid cross-contaminations, each virus treatment group was housed isolated in separate cages. AVs used for animal experiments were propagated, enriched and quantified as described in section 2.4. Virus quantities used are displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA.\u003c/p\u003e \u003cdiv id=\"Sec22\" class=\"Section3\"\u003e \u003ch2\u003e2.7.1 AVs treatment of nude mice bearing an induced G361 human melanoma\u003c/h2\u003e \u003cp\u003eHuman melanoma G361 cells were injected subcutaneously (s.c.) (1x10\u003csup\u003e6\u003c/sup\u003e cells/mice) into the right flank of 8-10-week-old female nude mice (Envigo, Israel). On day 10 after tumor induction, upon visualizing pulpable yet too small to be measured tumors, animals were randomly divided into four treatment groups and 100 \u0026micro;l of ARV-s1133, IBDV-WF, aMPV-P20 or PBS as control were intratumorally (i.t.) injected. On day 20, groups receiving viruses were injected with a second 100 \u0026micro;l dose containing a cocktail of all three viruses (1:1:1 v/v from the stock viral solutions following their propagation in VERO cells) and the control group received a second PBS dose. Tumor volumes were measured tri-weekly by caliper. Mice were euthanized upon tumor reaching maximal size in accordance with ethics regulation (12mm diameter).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003e2.7.2 AVs treatment of immunocompetent BALBc mice bearing an induced 4T1 mammary carcinoma\u003c/h2\u003e \u003cp\u003eMouse mammary cancer 4T1 cells were injected s.c. (5x10\u003csup\u003e4\u003c/sup\u003e cells/mice) into the right flank of 8-10-week-old female BALBc mice (Envigo, Israel). On day 5 after tumor induction, upon visualizing pulpable yet too small to be measured tumors, 6 mice per group were injected i.t. with 100 \u0026micro;l of ARV-s1133, IBDV-WF, aMPV-P20 or PBS as control. Treatments were administered again 7 and 14 days later. Blood samples were collected at baseline and 6 days after each treatment. Tumor volumes were measured tri-weekly by caliper. Mice were euthanized upon tumor reaching maximal size in accordance with ethics regulation (12mm diameter).\u003c/p\u003e \u003cp\u003e \u003cem\u003eAVs treatment of immunocompetent C57BL6 mice bearing an induced MC38 colon carcinoma\u003c/em\u003e \u003c/p\u003e \u003cp\u003eMouse colon cancer MC38 cells were injected s.c. (4x10\u003csup\u003e5\u003c/sup\u003e cells/mice) into the right flank of 8\u0026ndash;10 weeks old female C57BL6 mice (Envigo, Israel). On day 7 after tumor induction, upon visualizing pulpable yet too small to be measured tumors, 6 mice per group were injected i.t. with 100 \u0026micro;l of ARV-s1133, IBDV-WF, aMPV-P20 or PBS as control. Treatment was administered again 7 and 14 days later mice. Blood samples were collected at baseline and 6 days after each treatment. Tumor volumes were measured tri-weekly by caliper. Mice were euthanized upon tumor reaching maximal size in accordance with ethics regulation (12mm diameter).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section3\"\u003e \u003ch2\u003e2.7.3 Evaluation of anti-viral humoral immune response by ELISA\u003c/h2\u003e \u003cp\u003eBlood samples from treated mice were allowed to clot overnight at 4 \u0026ordm;C, after which, sera were separated. To assess the humoral immune responses raised against ARV s1133 and IBDV WF, 96-well ELISA plates (Thermo Fisher Scientific, USA) were coated overnight at 4 \u0026ordm;C with sigma C [16] or VP2 [17] polypeptides, respectively. Then, following washes and blocking (as specified in Section \u0026lrm;2.6.1), mouse sera diluted 1:100 were added and incubated 1 h at 37 \u0026ordm;C. After additional washes, plates were incubated 1 h at 37 \u0026ordm;C with goat anti-mouse peroxidase (Sigma, Israel), then washed, stained with OPD and absorbance was measured at 450 nm.\u003c/p\u003e \u003cp\u003eTo test responses raised against aMPV, a commercial ELISA kit (IDEXX Laboratories, USA) was used per manufacturer\u0026rsquo;s instructions, with the exception of substitution of the kits anti-chicken secondary antibodies with the above-mentioned anti-mouse antibodies. For technical reasons ELISA results for the determination of anti-aMPV antibody levels were not satisfactorily achieved.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Statistical analysis\u003c/h2\u003e \u003cp\u003eStatistical significance was assessed using student\u0026rsquo;s T test, analysis of variance (ANOVA) Tukey or ANOVA Dunnett test, as indicated in figure legends. Statistical analyses were performed using GraphPad Prism software.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 RESULTS","content":"\u003cdiv id=\"Sec27\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 Oncolytic activity of avian viruses (AVs) in vitro\u003c/h2\u003e\n \u003cp\u003eEight AVs, from four viral families, were evaluated \u003cem\u003ein vitro\u003c/em\u003e for their oncolytic potential. The AVs included two strains of ARV, three strains of IBDV and two strains of IBV and one strain of aMPV. Their effects were tested in the VERO cell line, FF normal human fibroblasts and 18 cancer cell lines from human or mouse origin (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). ARV strains s1133 and ISR5215, IBDV-WF and aMPV-P20 displayed effectively infected and damaged VERO cells and some cancer cell lines, but caused no apparent damage to the normal FF culture (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). While the attenuated ARV-s1133 vaccine strain caused various levels of damage to 14 out of the 18 cancer cell lines tested, the virulent ARV-ISR5215 field-isolated strain caused milder damage to five cell lines only (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA and Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Notably, all viruses used for this experiment were grown to their maximal potential in the VERO cells and used without further quantification. Therefore, viruses can be compared for their effect on distinct cell lines but not with each other. The aMPV-P20 strain affected 9 out of 18 cancer cell lines (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e and as demonstrated in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA and \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB). Of the three IBDV strains tested, only IBDV-WF mildly affected VERO cells, and induced acute damage to human melanoma cell lines M-15 and M-373, mild effect on G361 and moderate damage to mouse mammary (4T1) and renal (RenCa) cancer cell lines (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e and as demonstrated in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA and \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC). Both IBV strains tested imparted no visible cytopathic effect on any of the cells used in this study, except for a mild effect of IBV-M41 on LNCaP cells (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e), and therefore were excluded from further testing. Notably, the two ALL cell lines, REH and 697, seemed entirely resistant to all viruses, while all other cancer cell lines used were affected by at least one of the viruses tested (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eTable 1. Cytopathic effect of stock viruses assessed in cell lines\u003c/p\u003e\n \u003cp\u003e\u003cimg src=\"https://myfiles.space/user_files/69519_bce2c0439cd956a6/69519_custom_files/img1748978139.png\"\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003eAvian reovirus (ARV), Infectious bursal disease virus (IBDV) Infectious bronchitis virus (IBV), Avian Metapneumovirus (aMPV). Acute effect (+++), medium effect (++), mild effect (+), inconclusive, non-repeating effect (\u0026plusmn;), No apparent effect (-), not tested (nt). * Foreskin fibroblasts are primary culture.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 AV enrichment post-propagation in VERO cells\u003c/h2\u003e\n \u003cp\u003eThe three AVs inducing the most damage to cancer cell lines (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) namely, ARV-s1133, IBDV-WF and aMPV-P20, were propagated in large volumes in VERO cells and then enriched by ultracentrifugation. All three AVs displayed the ability to propagate in VERO cells, and TCID\u003csub\u003e50\u003c/sub\u003e/ml values of 6.4, 5 and 3 for ARV-s1133, IBDV-WF and aMPV-P20, respectively, were reached (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). TCID\u003csub\u003e50\u003c/sub\u003e/ml values after concentration enrichment were elevated by 5.6, 4.3 and 3.3 logs for ARV-s1133, IBDV-WF and aMPV-P20, respectively (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). AV activity and its increase post-concentration enrichment were also confirmed by reduction in the viability of VERO cells (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). Quantified viruses in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA were stored and later used in all mice experiments.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3 aMPV-P20 propagation in cancer cells\u003c/h2\u003e\n \u003cp\u003eTo evaluate the correlation between the virus-induced cytopathic effect and viral ability to propagate and release from cancer cells, aMPV was allowed to infect four cancer cell lines which were acutely (G361 and SW480), mildly (DU145) or not (MDA-KB2) affected by the virus (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Cell culture media samples containing extracellular aMPV-P20 which were released from infected cells were collected over 14 days and viral levels were quantified by ELISA. A significant increase in extracellular aMPV-P20 levels was detected over time in the SW480 culture, and reached levels similar to those measured in the control VERO cells (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). No increase was evident in MDA-KB2 and DU145 cells, for which a low or no cytopathic effect was observed after infection. Notably, no propagation was detected in the G361 melanoma cells despite the acute cytopathic effect induced by the virus (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). To further substantiate the ability of aMPV to propagate in the SW480 cell line, cell cultures were treated with ten-fold dilutions of the virus, and incubated for 7 days, after which, virus levels were quantified by ELISA and by TCID50/ml. A significant increase in virus levels and activity was detected both by ELISA and TCID50/ml (Figs. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC, respectively) in the presence of cells as compared to cell-free samples. TCID50/ml indicated that aMPV-P20 levels increased by more than 7 logs in the SW480 cells. Taken together, these results indicate that aMPV-P20 can propagate in the SW480 cell line while also inflicting a cytopathic effect.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec30\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4 AV influence on human melanoma cell G361 tumors in nude mice\u003c/h2\u003e\n \u003cp\u003eOut of all the AV strains tested \u003cem\u003ein vitro\u003c/em\u003e, ARV-s1133, aMPV-P20 and IBDV-WF displayed the widest ability to induce cytopathic and cancer-cell-killing effects (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e and Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) and were therefore chosen for further testing \u003cem\u003ein vivo.\u003c/em\u003e First, we evaluated the chosen AVs ability to induce tumor suppression on nude mice bearing a s.c. human melanoma G361 tumors. In a preliminary small scale study, mice were injected i.t. on day 10 after tumor inoculation with a single AV or PBS (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). Inhibition of tumor growth compared to the PBS control treatment was observed for all three AVs as soon as 7 days after the first treatment. On day 10 after the first treatment, the measured mean tumor volumes (\u0026plusmn;\u0026thinsp;SD) were 223.7 mm\u003csup\u003e3\u003c/sup\u003e (\u0026plusmn;\u0026thinsp;25.9), 169.7 mm\u003csup\u003e3\u003c/sup\u003e (\u0026plusmn;\u0026thinsp;36.1) and 196.8 mm\u003csup\u003e3\u003c/sup\u003e (\u0026plusmn;\u0026thinsp;80.3) for ARV-s1133, IBDV-WF and aMPV-P20 respectively, as compared to 434.1 mm\u003csup\u003e3\u003c/sup\u003e (\u0026plusmn;\u0026thinsp;607.6) for the PBS treatment (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB). These measurements signify a percent increase (\u0026plusmn;\u0026thinsp;SD) of 264.7% (\u0026plusmn;\u0026thinsp;12.9), 229.9% (\u0026plusmn;\u0026thinsp;107.9) and 174.3% \u0026plusmn; (18.6) for ARV-s1133, IBDV-WF and aMPV-P20 respectively, as compared to 1456.4% (\u0026plusmn;\u0026thinsp;1936.7) for the PBS treatment (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC). Of note, although virus treatments displayed substantial and unified inhibition of tumors progression, the effect was not statistically significant due to the high variability in the control group.\u003c/p\u003e\n \u003cp\u003eA second treatment was administered on day 20, with the PBS control group given a second PBS dose while all other groups were injected with a cocktail of all three AVs in order to test the combined viral effect on the tumors (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e\n \u003cp\u003eThe second dose of AVs significantly inhibited tumor progress in the mice treated with ARV-s1133 and aMPV-P20 as compared to the PBS-treated group while non statistically significant reduction was evident in the IBDV-WF treated group. A 341.5% (\u0026plusmn;\u0026thinsp;143.6) increase in tumor size was measured in the PBS treated group 20\u0026ndash;21 days (day 41) after administration of the second dose, while tumors in the ARV-s1133-, IBDV-WF- and aMPV-P20-treated groups increased by only 10.2% (\u0026plusmn;\u0026thinsp;67.6), 129.9% (\u0026plusmn;\u0026thinsp;95.7) and 79.4% (\u0026plusmn;\u0026thinsp;77.9), respectively (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec31\" class=\"Section2\"\u003e\n \u003ch2\u003e3.5 AV influence on murine mammary carcinoma 4T1 cells and tumors in immunocompetent BALBc mice\u003c/h2\u003e\n \u003cp\u003eAs the Achilles\u0026apos; heel of virotherapy is neutralization by the immune system, the ability of AVs to inhibit tumor growth in immunocompetent mice was also assessed.\u003c/p\u003e\n \u003cp\u003eMouse 4T1 mammary cells were similarly affected \u003cem\u003ein vitro\u003c/em\u003e by all three AVs and exhibited a concentration-dependent reduction in cell viability, reaching approximately 50% at the highest viral concentrations (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA). Notably, although acute (ARV-s1133) and medium (IBDV-WF, aMPV-P20) cytopathic effects were visually demonstrated (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e), a 10-fold dilution of each of the viruses seemed to diminish their influence on the cells in the cell viability assay (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e\n \u003cp\u003e4T1 cells were s.c. injected into BALBc mice. When tumors were palpable (on day 5) mice were i.t. administered either ARV-s1133, IBDV-WF, aMPV-P20 or PBS, followed by identical doses 7 and 14 days thereafter. A significant increase in the levels of antibodies raised against ARV-s1133 (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB) and IBDV-WF (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC) was detected after the second and third treatment doses, respectively. A significant decrease in tumor progression was seen in the animals treated with ARV-s1133 and IBDV-WF starting 2 days after administration of the second dose (day 14), while in the group treated with aMPV-P20, tumor progression resembled that of the PBS-treated negative control group (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec32\" class=\"Section2\"\u003e\n \u003ch2\u003e3.6 AV influence on murine colon carcinoma MC38 cells and tumors in immunocompetent C57BL/6 mice.\u003c/h2\u003e\n \u003cp\u003eTo further establish the potential of AVs, another cancer model was studied in immunocompetent C57BL/6 mice. MC38 cell viability was significantly lowered \u003cem\u003ein vitro\u003c/em\u003e when infected with the AVs (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA). ARV-s1133 and aMPV-P20 enrichment significantly increased viral potency as reflected by a reduction in MC38 cell viability, while IBDV-WF displayed similar activity before and after enrichment (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA). MC38 cells were s.c injected into C57BL/6 mice. When tumors were palpable (day 7), ARV-s1133, IBDV-WF, aMPV-P20 or PBS was i.t. injected, and second and third doses were administered 7 and 14 days thereafter. Similar to the observed response in the 4T1-BALBc model, a significant increase in the serum antibodies raised against ARV-s1133 (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB) and IBDV-WF (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC) was detected in the MC38-C57BL6 model after the second and third treatment dose, respectively. Although not significant, a trend of decrease in tumor progression was already evident on the day of the second IBDV-WF and aMPV-P20 treatment dose (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eD). Notably, ARV-s1133 which was very potent in inhibiting 4T1 tumor progression \u003cem\u003ein vivo\u003c/em\u003e and reducing both 4T1 and MC38 cell viability \u003cem\u003ein vitro\u003c/em\u003e, displayed only a mild inhibitory effect on the MC38 tumors. Corresponding to tumor progression, mouse survival was significantly prolonged in the aMPV-P20-treated group as compared to the PBS-treated group. Survival of IBDV-WF-treated mice was also extended, but not significantly (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eE).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eOVs have gained considerable attention as a potential therapeutic modality for cancer treatment due to their ability to selectively target and destroy cancer cells while sparing normal cells [18]. Various natural or attenuated as well as genetically modified OVs from human or animal origin, including adenoviruses, herpes simplex viruses and reoviruses, have been tested in preclinical and clinical studies [18]. Clinical trials have demonstrated the potential of OVs as cancer therapeutics for the treatment of melanoma [19], breast cancer [20], ovarian cancer [21] and others [22].\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003eIn this study, viruses of avian origin were tested to identify those that can selectively destroy cancer cells of mouse and human origin. Out of the eight tested AVs of four different viral families, four viruses induced acute cytopathic effects in one or more cancer cell lines, while normal fibroblasts remained intact. Several mechanisms of action determining susceptibility versus resistance of cells to OVs were described e.g. lack of PKR related RNA degradation due to overactivation of Kras. The differences in susceptibility of several cell lines seen in this study may be explained by presence or lack of such mechanisms relevant to the viruses used in this study. Out of the two ARV strains that induced a cytopathic effect in cancer cells, the attenuated strain s1133 induced a cytopathic effect in 14 of the 18 cancer cell lines tested, while the virulent ISR5215 strain caused milder damage to five lines only. Similarly, the attenuated vaccine IBDV-WF strain was superior to the virulent strain in inducing a cytopathic effect in two human melanoma cell lines and lung and breast cancer mice cell lines. These findings indicate that virus virulence in poultry does not correlate with their effects on cancer cells in mammals.\u003c/p\u003e \u003cp\u003eKim et al. demonstrated that prolonged exposure of human HT1080 fibrosarcoma cells to reovirus yielded high levels of virus-resistant cells [23]. The variety of potential OVs available, that differ in their origin, and possibly possessing different mechanisms of action, and the possibility of employing combinations of viruses as cocktails or in succession, may successfully circumvent development of cancer resistance during treatment.\u003c/p\u003e \u003cp\u003eThe ARV-s1133, IBDV-WF and aMPV-P20 viruses, which displayed the most pronounced effects on cancer cells, were further evaluated \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e for their potential as OV. Methods for viral propagation and enrichment were developed for each individual AV. Propagation of aMPV in SW480 colon cancer cell line, as established in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and affirmed in Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, was accompanied by an acute virus-induced cytopathic effect. In MDA-Kb2 cells, neither a cytopathic effect nor viral propagation were documented. While viral propagation was expected to contribute to cell destruction, aMPV induced a mild and acute viral cytopathic effect in DU145 and G361 cell lines, respectively, despite their failure to propagate in these cells. This could indicate that other mechanisms for virus-induced cell destruction, such as apoptosis or others are also in play, and may contribute to the anti-cancer viral activities of the AVs.\u003c/p\u003e \u003cp\u003eThree mouse models were used to evaluate the oncolytic effects of AVs. The oncolytic effect of the viruses was first evaluated in nude mice carrying G361 human melanoma tumors. The absence of a fully functional acquired immune system in this model enabled assessment of the influence of AVs on human cancers. Differences between the effect of all three AVs on tumors and the PBS negative control were already observed after the first dose. These differences increased and became statistically significant following administration, on day 20 following tumor induction, of the second treatment dose of AVs cocktail in the groups readministered with the aMPV-P20, and the ARV-s1133. Similar tumor inhibition trends were seen for all three AVs treated groups. The contribution of each AV effect on tumor inhibition in this experiment was evaluated only after the first dose. Following administration of the cocktail as a second dose, the effect shown may result from the combined activity of all 3 AVs. The effect of each single AV on tumor inhibition was evaluated and demonstrated in later experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eThe ability of viral treatments to retain their effectiveness in parallel to the development of a debilitating targeted immune response was tested in immunocompetent BALBc and C57BL6 mice carrying 4T1 mammary and MC38 colon cancers, respectively. These cells were selected for testing \u003cem\u003ein vivo\u003c/em\u003e after their observed susceptibility to AVs \u003cem\u003ein vitro\u003c/em\u003e. Antibodies toward ARV-s1133 and IBDV-WF were detected following the second and third intra-tumoral virus doses, respectively. Elevation of antibody levels was timed closely with increased tumor progression rate in AV treated mice and may explain the decreased efficacy of their oncolytic effect.\u003c/p\u003e \u003cp\u003eThe immune response toward OVs has two faces. On the one hand, the local immune response toward the virus and the infected cancer cells may activate an anti-tumor immune response in the tumor microenvironment [24,25]. This may enhance the efficacy of treatment, since, in addition to oncolysis, the tumor-specific immune response is raised due to presence of viral epitopes and induction of innate and cytotoxic T cell immunity in the tumor microenvironment. This response may add to the destruction of tumor cells, and initiate an immune response against metastasis [26]. On the other hand, after several doses, humoral immune response may neutralize the virus.Pre-existing or newly developed neutralizing antibodies recognizing the virus may disable the treatment over multiple dosing [27,28]. For example, Russell et al. reported that when attempting to use an attenuated measles virus vaccine strain engineered to target CD46 that is abundant on human tumor cells, the major impediment to the treatment efficacy was the pre-existing anti-measles immunity [28]. One way to overcome the immune response is by direct injection of the virus into the tumors [29], which prevents spillover of the vector to other tissues. A possible disadvantage of this approach is the probable limitation of the treatment to the tumor, which may not enable treatment of metastases. Viral migration from the injected tumor site may elicit neutralizing antibodies which will limit treatment effectiveness to the metastasis. A combined treatment of oncolytic virus with immuno-suppressive drugs was suggested to overcome the immune response and to induce metastatic tumor regression [30].\u003c/p\u003e \u003cp\u003eIn the current study, three viruses from three different families, were found to induce tumor cell destruction \u003cem\u003ein vitro\u003c/em\u003e. In addition to the ARV-s1133 vaccine strain, a field isolate was also tested, and its ability to kill cancer cells \u003cem\u003ein vitro\u003c/em\u003e was demonstrated. The genetic variation of ARV is determined by the sequence of sigma C. This protein induces the production of neutralizing antibodies, and its variation enables the virus to escape the immune response [31]. The high variability of ARV generates different strains that evade neutralizing antibodies raised against other strains [32,33], which ultimately highly enriches the arsenal of viruses that can be used for treatment. Induction of an immune response against the tumor while enabling continuation of the treatment by switching the treatment viruses, allows for combined and prolonged tumor attack both by the virus and the immune system.\u003c/p\u003e \u003cp\u003eThe growth of 4T1 tumors was significantly inhibited following treatment with ARV-s1133 and IBDV-WF. The growth of colon cancer cell line MC38 tumors was significantly inhibited and survival of mice bearing s.c. MC38 tumors was extended following treatment with aMPV-P20 and IBDV-WF. Following repeated AV administrations, tumors began to re-grow in correlation with specific anti-virus antibody titers. A possible route to overcome decrease of treatment efficacy due to the rise of the neutralizing immune response may be to simultaneously or sequentially administer other OVs.\u003c/p\u003e \u003cp\u003eNo clinical adverse effects, including body weight loss or abnormal appearance and behavior, were observed as result of virus administration in any of the tested mouse models (data not shown). These viruses are non-zoonotic and are widely used as commercial live attenuated vaccines in poultry. No evidence of adverse risk for farmers who are constantly exposed to those viruses was reported.\u003c/p\u003e \u003cp\u003eIn conclusion, this study characterized the oncolytic potential of three AVs. The AVs induced cytopathic effects and effectively inhibited cancer cell line proliferation \u003cem\u003ein vitro\u003c/em\u003e and delayed tumor growth \u003cem\u003ein vivo\u003c/em\u003e. The AVs along with their immunogenetically distinct field variants, bear potential to serve as a vast arsenal of OVs to enable prolonged and effective treatment of susceptible cancers.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eEthics statement\u003c/h2\u003e \u003cp\u003eAnimal experiments were conducted with the approval of the Israeli ethics committee (ethic numbers IL-19-5-251 and IL-19-2-98).\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThis research was funded internally by MIGAL institute.\u003c/p\u003e\u003ch2\u003eACKNOWLEDGMENT.\u003c/h2\u003e \u003cp\u003eThe authors would like to thank Prof. Jamal Mahajna from MIGAL institute in the Galilee for his support, and Prof. Michal Lotem from Hadassah Medical Center in Jerusalem for melanoma cells.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eJin, K.-T.; Du, W.-L.; Liu, Y.-Y.; Lan, H.-R.; Si, J.-X.; Mou, X.-Z.; Marchini, A.; Ilkow, C.S.; Melcher, A. 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Wide-Range Protection against Avian Reovirus Conferred by Vaccination with Representatives of Four Defined Genotypes. \u003cem\u003eVaccine\u003c/em\u003e\u003cstrong\u003e2011\u003c/strong\u003e, \u003cem\u003e29\u003c/em\u003e, 8683\u0026ndash;8688, doi:10.1016/J.VACCINE.2011.08.114.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"archives-of-virology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"arvi","sideBox":"Learn more about [Archives of Virology](https://www.springer.com/journal/705)","snPcode":"705","submissionUrl":"https://submission.nature.com/new-submission/705/3","title":"Archives of Virology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Oncolytic viruses, Cancer virotherapy, Avian reovirus, Infectious bursal disease virus, Avian metapneumovirus","lastPublishedDoi":"10.21203/rs.3.rs-6690670/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6690670/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOncolytic viruses (OVs) can selectively infect and kill tumor cells. Although showing promise, several challenges impede OVs broad application in cancer therapy. A major obstacle is limited treatment duration due to preexisting or the induction of neutralizing immune responses toward the OVs following treatment. Widening the reservoir of OVs will allow replacement of treatment viruses following neutralization. This study aimed to identify new OVs and to test their oncolytic effect. Eight avian viruses (AVs) were used to infect human and mice normal and cancerous cell lines, of which three displayed superior ability to selectively kill cancer cells \u003cem\u003ein vitro.\u003c/em\u003e These AVs induced cytopathic effects and inhibited proliferation of fifteen out of eighteen cancer cell lines tested, each affecting 5\u0026ndash;14 of the cell lines; none affected normal fibroblasts. \u003cem\u003eIn vivo\u003c/em\u003e, growth of G361 melanoma cell tumors in nude mice was inhibited following intra-tumoral (i.t) injection of AVs. In two models of immunocompetent mice carrying tumors and injected i.t with AVs, tumors growth was significantly delayed. Albeit tumor growth commenced in correlation with the development of anti-virus antibody levels. These tested AVs together with their field-characterized variants, comprise a vast arsenal of potential OVs that may open the possibility of administration of several viruses in mix or in sequence to overcome both tumor resistance due to acquired mutations as well as neutralization by the rise of the acquired immune response. In conclusion, the AVs tested in this study demonstrated OVs characteristics and may be used to enable prolonged cancer virotherapy treatment.\u003c/p\u003e","manuscriptTitle":"Viruses of Avian Origin for Cancer Virotherapy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-03 19:20:58","doi":"10.21203/rs.3.rs-6690670/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revision","date":"2025-08-09T20:52:08+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-06-04T05:31:56+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-27T19:33:05+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-22T03:24:43+00:00","index":"","fulltext":""},{"type":"submitted","content":"Archives of Virology","date":"2025-05-18T04:51:28+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"archives-of-virology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"arvi","sideBox":"Learn more about [Archives of Virology](https://www.springer.com/journal/705)","snPcode":"705","submissionUrl":"https://submission.nature.com/new-submission/705/3","title":"Archives of Virology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a42d3b1b-4f3f-464e-bf80-68d2b5fe1462","owner":[],"postedDate":"June 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-23T16:12:59+00:00","versionOfRecord":{"articleIdentity":"rs-6690670","link":"https://doi.org/10.1007/s00705-026-06559-8","journal":{"identity":"archives-of-virology","isVorOnly":false,"title":"Archives of Virology"},"publishedOn":"2026-02-21 15:58:53","publishedOnDateReadable":"February 21st, 2026"},"versionCreatedAt":"2025-06-03 19:20:58","video":"","vorDoi":"10.1007/s00705-026-06559-8","vorDoiUrl":"https://doi.org/10.1007/s00705-026-06559-8","workflowStages":[]},"version":"v1","identity":"rs-6690670","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6690670","identity":"rs-6690670","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}