Photodynamic inactivation of selected bovine viruses by isomeric cationic tetra-platinated porphyrins

Porphyrin-based photodynamic processes have been used for the inactivation of microorganisms and treatment of tumors. The virucidal activity of porphyrins 3-PtTPyP and 4-PtTPyP was investigated against bovine viruses representative of the main structural groups (enveloped/non-enveloped, DNA/RNA: BVDV, BoHV-1, BAV and BEV), and against two epitheliotropic viruses (VSV and VACV). Viral suspensions were incubated at 0.91 [Formula: see text]mol [Formula: see text] L[Formula: see text] and exposed to a white-light LED array source (25 mW [Formula: see text] cm[Formula: see text]; 90 J [Formula: see text] cm[Formula: see text] for 0, 15, 30 and 60 min followed by determination of the remaining virus titers. Porphyrin 3-PtTPyP reduced almost 6 log of VSV and 3.5 log of BVDV titers after 15 min and complete virus photoinactivation was achieved after 30 min. 4-PtTPyP at 0.91 [Formula: see text]mol [Formula: see text] L[Formula: see text] produced reduction of titers of all enveloped virus depending on the time of light irradiation. No virucidal activity of any of the porphyrins was observed for non-enveloped viruses and these results showed the potential of porphyrins to inactivate viruses in premises.

Oncolytic Virotherapy in Multiple Myeloma: A Possible Alternative Role of Bovine Viruses.

Multiple Myeloma (MM) is a plasma cell malignancy characterized by the tight dependence to the bone marrow (BM) microenvironment that supports MM cells survival. Despite significant therapeutic progress in the recent years with the introduction of several new drugs, MM remains an incurable disease. Oncolytic virotherapy is an alternative therapeutic technology in the cancer treatment exploiting naturally or genetically engineered viruses able to infect, transduce and consequently kill cancer cells directly or indirectly through the delivery of the microenvironment cells. Several oncolytic viruses have shown promising pre-clinical results for the treatment of MM, in particular Measles virus and Reovirus. However, the use of human viruses such as Measles could be limited by the antiviral immune response of the patients due to vaccination or natural infection. In order to avoid these potential limits of the human viruses, the aim of this study was to investigate the development of bovine viruses as an alternative oncolytic strategy in MM by checking these viruses that showed an anti-tumoral activity in different solid tumors. Thus, we investigated the potential lytic effect on human MM cells of the Bovine Viral Diarrhea Virus (BVDV), known to bind CD46 as reported for human measles virus, and the Oncolytic Bovine Herpesvirus type 4 (BoHV-4). Firstly, we treated several human myeloma cell lines (HMCLs) with BVDV or the heat-inactivated virus for 24, 48 and 72 hours. The infection efficiency was verified by nested multiplex PCR. We showed a significant increase of cell mortality, checked by trypan blue count and flow cytometry analysis, already after 48 hours of infection in JJN3 (mean±SD % of dead cells: BVDV 45±11 % vs inactive virus 16±2.5 %, p=0.013), and in OPM2 (BVDV 43±1.4 % vs inactive virus 28±2.1 %, p= 0.015) but not in U266 (BVDV 25±23 % vs inactive virus 18±12 %, p=NS. However, BVDV pre-treatment for 12 hours and followed by 48 hours bortezomib (bor) treatment (concentration ranging: 5-10nM) significantly restored bor sensitivity in U266 resistant cells (mean±SD % of dead cells: BVDV plus bor 10 nM 69±8 % vs inactive virus + bor 10 nM 36±1 %, p= 0.031). Interestingly, the cytotoxic effect of BVDV treatment in HMCLs was associated by a significantly increase of apoptotic markers evaluated by flow cytometry. Subsequently, we infected BM primary purified CD138+, showing a significant increase of the mortality rate after treatment with BVDV as compared to the inactivated virus. On the contrary, BVDV was not able to infect human BM mesenchymal stromal cells (MSCs) not showing any lytic effect. Thereafter the capacity to induce MM cell lysis by a recombinant BoHV-4 virus, delivering a Red Fluorescent Protein (RFP) expression cassette as reporter gene, was also evaluated. As observed by the percentage of RFP-positive cells, BoHV-4 was unable to infect and consequently to kill several HMCLs tested. Then we used BM MSCs as in vitro model for oncolytic virus delivery in co-culture systems with MM cells. BoHV-4 infected hTERT-MSCs, expressing RFP at 24, 48 and 72 hours. Consistently, hTERT-MSCs viability was progressively reduced at 24 and 48 hours after infection, as compared to controls, (mean±SD % reduction of cell viability: -22±8 %, p=0.0254 and -49±2 %, p=0.0001, respectively), reaching the highest effect at 72 hours (-70±1.5 %, p=0.0003). Thus we evaluated the effect of BoHV-4 in a co-culture system between human MSCs and two stroma-dependent HMCLs as INA-6 and saMMi. In both cases the percentage of dead HMCLs increased in co-culture with BoHV-4 infected hTERT-MSCs, as compared to hTERT-MSCs untreated controls (INA-6: BoHV-4 61±2.1 % vs control 12±2.1 %, p= 0.0018; saMMi: BoHV-4 48±1.9 % vs control 14±1.4 %, p= 0.0027). Overall our data indicate both direct and MSC-mediated oncolytic effects of bovine viruses on MM cell, suggesting their possible use as novel alternative anti-MM virotherapy strategy. Disclosures Giuliani: Celgene: Research Funding; Janssen: Research Funding.

Integrin-Using Rotaviruses Bind α2β1 Integrin α2 I Domain via VP4 DGE Sequence and Recognize αXβ2 and αVβ3 by Using VP7 during Cell Entry

ABSTRACT Integrins α2β1, αXβ2, and αVβ3 have been implicated in rotavirus cell attachment and entry. The virus spike protein VP4 contains the α2β1 ligand sequence DGE at amino acid positions 308 to 310, and the outer capsid protein VP7 contains the αXβ2 ligand sequence GPR. To determine the viral proteins and sequences involved and to define the roles of α2β1, αXβ2, and αVβ3, we analyzed the ability of rotaviruses and their reassortants to use these integrins for cell binding and infection and the effect of peptides DGEA and GPRP on these events. Many laboratory-adapted human, monkey, and bovine viruses used integrins, whereas all porcine viruses were integrin independent. The integrin-using rotavirus strains each interacted with all three integrins. Integrin usage related to VP4 serotype independently of sialic acid usage. Analysis of rotavirus reassortants and assays of virus binding and infectivity in integrin-transfected cells showed that VP4 bound α2β1, and VP7 interacted with αXβ2 and αVβ3 at a postbinding stage. DGEA inhibited rotavirus binding to α2β1 and infectivity, whereas GPRP binding to αXβ2 inhibited infectivity but not binding. The truncated VP5* subunit of VP4, expressed as a glutathione S-transferase fusion protein, bound the expressed α2 I domain. Alanine mutagenesis of D308 and G309 in VP5* eliminated VP5* binding to the α2 I domain. In a novel process, integrin-using viruses bind the α2 I domain of α2β1 via DGE in VP4 and interact with αXβ2 (via GPR) and αVβ3 by using VP7 to facilitate cell entry and infection.