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Bovine herpesvirus 4 based vector as a potential oncolytic-virus for treatment of glioma.

Redaelli M, Mucignat-Caretta C, Cavaggioni A, Caretta A, D'Avella D, Denaro L, Cavirani S, Donofrio G - Virol. J. (2010)

Bottom Line: Here, we investigated bovine herpesvirus 4 (BoHV-4), which is non-pathogenic if injected into the rodent brain.BoHV-4 was injected into a tumour grown in rat brain.Although virus expression was scattered across the tumour mass, it was mainly located in the peripheral area of larger gliomas.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Animal Health, University of Parma, Italy.

ABSTRACT
The application of gene therapy for malignant gliomas is still under study and the use of specific vectors represents an important contribution. Here, we investigated bovine herpesvirus 4 (BoHV-4), which is non-pathogenic if injected into the rodent brain. We show that the vector can infect mouse, rat and human glioma cell lines and primary cultures obtained from human glioblastoma in vitro. BoHV-4 was injected into a tumour grown in rat brain. Although virus expression was scattered across the tumour mass, it was mainly located in the peripheral area of larger gliomas. These data support BoHV-4 as a candidate vector for glioma treatment.

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Representative pictures (10×) of BoHV-4-EFGPΔTK infected F98 (A), GLI36 (C) and GL261 (E) cells at 96 hours (hs) post infection (P.I.), visualized by phase contrast (PC) fluorescence with a FITC filter for EGFP expression or with DAPI filter for nuclear counterstaining (bar = 100 μm). The respective titers (expressed as log10 of Tissue Cells Infectious Dose/50 [TCID50] per ml-1) of viral particles released during the time at 24 and 96 hours (hs) post infection (P.I.) are shown in B, D and F. Values are the mean ± standard error of three independent experiments. (G) GL261 mouse glioblastoma cell line (a, bar = 25 μm), F98 rat glioma cell line (b, bar = 25 μm) and GLI36 human glioma cell line (c, bar = 10 μm) infected with BoHV-4EGFPΔTK for 72 hours. CPE induced by infection shows a prevalence of necrosis (ANOVA, ** p < 0.001, *p < 0.05).
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Figure 1: Representative pictures (10×) of BoHV-4-EFGPΔTK infected F98 (A), GLI36 (C) and GL261 (E) cells at 96 hours (hs) post infection (P.I.), visualized by phase contrast (PC) fluorescence with a FITC filter for EGFP expression or with DAPI filter for nuclear counterstaining (bar = 100 μm). The respective titers (expressed as log10 of Tissue Cells Infectious Dose/50 [TCID50] per ml-1) of viral particles released during the time at 24 and 96 hours (hs) post infection (P.I.) are shown in B, D and F. Values are the mean ± standard error of three independent experiments. (G) GL261 mouse glioblastoma cell line (a, bar = 25 μm), F98 rat glioma cell line (b, bar = 25 μm) and GLI36 human glioma cell line (c, bar = 10 μm) infected with BoHV-4EGFPΔTK for 72 hours. CPE induced by infection shows a prevalence of necrosis (ANOVA, ** p < 0.001, *p < 0.05).

Mentions: Infection was performed with 1 TCID50/cell of a recombinant BoHV-4 expressing EGFP (BoHV-4EGFPΔTK) [3] and its effects were observed after 24, 48, 72, 96, 144, 216 hours post infection with an epi-fluorescence microscope (Leica). Indeed BoHV-4EGFPΔTK infected, replicated and induced cytopathic effects (CPE) in all three cell lines tested (Figure 1A, C and 1E). To quantify the newly produced progeny virus, the non-penetrated infectious viral particles were inactivated by low-pH treatment after infection. Cultures were washed with medium and cultured until CPE appeared, after which 1 ml of the medium was removed from each well and centrifuged for 5 min at 3000 rpm in a bench top centrifuge to remove any cellular debris and TCID50 were determined (tittering was repeated three times for each cell line). All three cell lines sustained productive infection (Figure 1B, D and 1F). In order to analyze the CPE induced by BoHV-4EGFPΔTK, cells were fixed with methanol and stained with Wright's stain. A total of 600 cells were counted from each slide, and the percentage of apoptotic and necrotic cells was calculated. At least 6 control and 6 treated slides were counted for each treatment. Monovariate ANOVA was used to test differences in the percentage of dead cells between control and infected cells. The CPE induced in vitro by BoHV-4EGFPΔTK infection was prevalently necrosis (Figure 1G) rather than apoptosis. Similar results were obtained with Annexin V and Propidium Iodide staining (data not shown). These results, together with the data previously obtained in vivo where BoHV-4 did not replicate in the mouse and rat brain, but reporter gene expression was shown following injection into the mouse and rat lateral ventricle, prompted us to investigate the use of BoHV-4 as a vector for the gene therapy or as an oncolytic virus of brain tumours. Thus, a rat glioma model was constructed. Fifteen four-month-old, male Fisher rats were pre-anesthetized with isoflurane and subsequently anesthetized with zolazepam tiletamine (20 mg/kg body weight) and xylazine (75 mg/kg body weight). Eight × 106 F98 glioma cells were suspended in 8 μl DMEM and injected 1 mm anterior and 1.5 mm lateral to the bregma, 3.7 mm below the pial surface. Injection was carried out for 16 minutes and was performed using a Hamilton syringe. Animals were monitored daily for neurological signs and weight loss. At the appearance of neurological signs, animals were re-anesthetized as above and 6 μl of 106 pfu of BoHV-4EGFPΔTK were injected into the same position as the previous injection. Animals were then monitored every 12 hours. Any animals showing severe worsening of neurological conditions were humanely euthanize. Rat brains were analyzed at different post-injection times: 48, 72, 86, 96, 120, 132, 144 and 216 hours. Briefly, anesthetized rats were first perfused with PBS for 15 min and then with 4% formalin in PBS for 30 min. Brains were carefully removed, post-fixed for 2 hours in 4% formalin in PBS, equilibrated for 24 h in 30% sucrose in PBS at 4°C and frozen at -80°C until sectioning with a cryostat at 16 μm. Sections of the BoHV-4-injected, rat brain gliomas showed EGFP expression in the peripheral area of larger tumours (Figure 2a, b), scattered across the mass of smaller tumours (Figure 2c), and in the solid peripheral area of cystic tumours (Figure 2d). These same sections, following observation of EGFP expression, were then stained with hematoxylin-eosin. In order to confirm co-localization of the tumour area with EGFP-positive transduced cells, five four-month-old male rats were inoculated with 8 × 106 F98 glioma cells labelled with the red fluorescent cell linker PHK26, according to manufacturer's instructions (Sigma). Cells maintain fluorescence for more than 100 mitotic divisions [9]. When BoHV-4EGFPΔTK was injected into the rat brains at the same position as the marked glioma cells, co-localization between the red fluorescent-marked tumour area and the EGFP positive cells was observed, without detection of the EGFP signal within the brain parenchyma (data not shown). In another experiment, primary cultures from biopsies of 2 patients with glioblastoma (both males, 59 and 79 years of age respectively) were prepared. Specimens were dissociated not more than 30 minutes after surgery by shaking for 5 minutes in 0.25% Trypsin, 0.02% EDTA (1 ml/mm3 tissue). The suspension was inactivated with CGM, and centrifuged at 37°C for 10 minutes at 1350 rpm. The supernatant was discharged and the pellet resuspended in 10 ml of CGM, changed every 72 hour for three weeks. Cells were then infected with BoHV-4EGFPΔTK and analyzed 24, 48 and 72 hours post infection as described above. Indeed, these primary cultures of human glioblastoma were susceptible to BoHV-4 infection as shown by EGFP expression, and also in this case infection leaded to a mainly necrotic CPE (Figure 3).


Bovine herpesvirus 4 based vector as a potential oncolytic-virus for treatment of glioma.

Redaelli M, Mucignat-Caretta C, Cavaggioni A, Caretta A, D'Avella D, Denaro L, Cavirani S, Donofrio G - Virol. J. (2010)

Representative pictures (10×) of BoHV-4-EFGPΔTK infected F98 (A), GLI36 (C) and GL261 (E) cells at 96 hours (hs) post infection (P.I.), visualized by phase contrast (PC) fluorescence with a FITC filter for EGFP expression or with DAPI filter for nuclear counterstaining (bar = 100 μm). The respective titers (expressed as log10 of Tissue Cells Infectious Dose/50 [TCID50] per ml-1) of viral particles released during the time at 24 and 96 hours (hs) post infection (P.I.) are shown in B, D and F. Values are the mean ± standard error of three independent experiments. (G) GL261 mouse glioblastoma cell line (a, bar = 25 μm), F98 rat glioma cell line (b, bar = 25 μm) and GLI36 human glioma cell line (c, bar = 10 μm) infected with BoHV-4EGFPΔTK for 72 hours. CPE induced by infection shows a prevalence of necrosis (ANOVA, ** p < 0.001, *p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2984596&req=5

Figure 1: Representative pictures (10×) of BoHV-4-EFGPΔTK infected F98 (A), GLI36 (C) and GL261 (E) cells at 96 hours (hs) post infection (P.I.), visualized by phase contrast (PC) fluorescence with a FITC filter for EGFP expression or with DAPI filter for nuclear counterstaining (bar = 100 μm). The respective titers (expressed as log10 of Tissue Cells Infectious Dose/50 [TCID50] per ml-1) of viral particles released during the time at 24 and 96 hours (hs) post infection (P.I.) are shown in B, D and F. Values are the mean ± standard error of three independent experiments. (G) GL261 mouse glioblastoma cell line (a, bar = 25 μm), F98 rat glioma cell line (b, bar = 25 μm) and GLI36 human glioma cell line (c, bar = 10 μm) infected with BoHV-4EGFPΔTK for 72 hours. CPE induced by infection shows a prevalence of necrosis (ANOVA, ** p < 0.001, *p < 0.05).
Mentions: Infection was performed with 1 TCID50/cell of a recombinant BoHV-4 expressing EGFP (BoHV-4EGFPΔTK) [3] and its effects were observed after 24, 48, 72, 96, 144, 216 hours post infection with an epi-fluorescence microscope (Leica). Indeed BoHV-4EGFPΔTK infected, replicated and induced cytopathic effects (CPE) in all three cell lines tested (Figure 1A, C and 1E). To quantify the newly produced progeny virus, the non-penetrated infectious viral particles were inactivated by low-pH treatment after infection. Cultures were washed with medium and cultured until CPE appeared, after which 1 ml of the medium was removed from each well and centrifuged for 5 min at 3000 rpm in a bench top centrifuge to remove any cellular debris and TCID50 were determined (tittering was repeated three times for each cell line). All three cell lines sustained productive infection (Figure 1B, D and 1F). In order to analyze the CPE induced by BoHV-4EGFPΔTK, cells were fixed with methanol and stained with Wright's stain. A total of 600 cells were counted from each slide, and the percentage of apoptotic and necrotic cells was calculated. At least 6 control and 6 treated slides were counted for each treatment. Monovariate ANOVA was used to test differences in the percentage of dead cells between control and infected cells. The CPE induced in vitro by BoHV-4EGFPΔTK infection was prevalently necrosis (Figure 1G) rather than apoptosis. Similar results were obtained with Annexin V and Propidium Iodide staining (data not shown). These results, together with the data previously obtained in vivo where BoHV-4 did not replicate in the mouse and rat brain, but reporter gene expression was shown following injection into the mouse and rat lateral ventricle, prompted us to investigate the use of BoHV-4 as a vector for the gene therapy or as an oncolytic virus of brain tumours. Thus, a rat glioma model was constructed. Fifteen four-month-old, male Fisher rats were pre-anesthetized with isoflurane and subsequently anesthetized with zolazepam tiletamine (20 mg/kg body weight) and xylazine (75 mg/kg body weight). Eight × 106 F98 glioma cells were suspended in 8 μl DMEM and injected 1 mm anterior and 1.5 mm lateral to the bregma, 3.7 mm below the pial surface. Injection was carried out for 16 minutes and was performed using a Hamilton syringe. Animals were monitored daily for neurological signs and weight loss. At the appearance of neurological signs, animals were re-anesthetized as above and 6 μl of 106 pfu of BoHV-4EGFPΔTK were injected into the same position as the previous injection. Animals were then monitored every 12 hours. Any animals showing severe worsening of neurological conditions were humanely euthanize. Rat brains were analyzed at different post-injection times: 48, 72, 86, 96, 120, 132, 144 and 216 hours. Briefly, anesthetized rats were first perfused with PBS for 15 min and then with 4% formalin in PBS for 30 min. Brains were carefully removed, post-fixed for 2 hours in 4% formalin in PBS, equilibrated for 24 h in 30% sucrose in PBS at 4°C and frozen at -80°C until sectioning with a cryostat at 16 μm. Sections of the BoHV-4-injected, rat brain gliomas showed EGFP expression in the peripheral area of larger tumours (Figure 2a, b), scattered across the mass of smaller tumours (Figure 2c), and in the solid peripheral area of cystic tumours (Figure 2d). These same sections, following observation of EGFP expression, were then stained with hematoxylin-eosin. In order to confirm co-localization of the tumour area with EGFP-positive transduced cells, five four-month-old male rats were inoculated with 8 × 106 F98 glioma cells labelled with the red fluorescent cell linker PHK26, according to manufacturer's instructions (Sigma). Cells maintain fluorescence for more than 100 mitotic divisions [9]. When BoHV-4EGFPΔTK was injected into the rat brains at the same position as the marked glioma cells, co-localization between the red fluorescent-marked tumour area and the EGFP positive cells was observed, without detection of the EGFP signal within the brain parenchyma (data not shown). In another experiment, primary cultures from biopsies of 2 patients with glioblastoma (both males, 59 and 79 years of age respectively) were prepared. Specimens were dissociated not more than 30 minutes after surgery by shaking for 5 minutes in 0.25% Trypsin, 0.02% EDTA (1 ml/mm3 tissue). The suspension was inactivated with CGM, and centrifuged at 37°C for 10 minutes at 1350 rpm. The supernatant was discharged and the pellet resuspended in 10 ml of CGM, changed every 72 hour for three weeks. Cells were then infected with BoHV-4EGFPΔTK and analyzed 24, 48 and 72 hours post infection as described above. Indeed, these primary cultures of human glioblastoma were susceptible to BoHV-4 infection as shown by EGFP expression, and also in this case infection leaded to a mainly necrotic CPE (Figure 3).

Bottom Line: Here, we investigated bovine herpesvirus 4 (BoHV-4), which is non-pathogenic if injected into the rodent brain.BoHV-4 was injected into a tumour grown in rat brain.Although virus expression was scattered across the tumour mass, it was mainly located in the peripheral area of larger gliomas.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Animal Health, University of Parma, Italy.

ABSTRACT
The application of gene therapy for malignant gliomas is still under study and the use of specific vectors represents an important contribution. Here, we investigated bovine herpesvirus 4 (BoHV-4), which is non-pathogenic if injected into the rodent brain. We show that the vector can infect mouse, rat and human glioma cell lines and primary cultures obtained from human glioblastoma in vitro. BoHV-4 was injected into a tumour grown in rat brain. Although virus expression was scattered across the tumour mass, it was mainly located in the peripheral area of larger gliomas. These data support BoHV-4 as a candidate vector for glioma treatment.

Show MeSH
Related in: MedlinePlus