Despite encouraging progress in the field of oncolytic viral therapy, several limitations remain in the development of an ideal OV including selective targeting of OVs to tumor tissue, relatively poor virus-spread throughout solid tumor tissue, inefficient viral replication in immune-competent hosts and disadvantageous ratio between anti-viral and anti-tumoral immunity. Several strategies to overcome these limitations are currently being investigated. The major strategies include the improvement of existing oncolytic vector systems or using combinations of oncolytic viruses with existing clinical methods or agents for synergistic and/or additive anti-tumor responses. Oncolytic virotherapy for canine cancers is still in its infancy; however, exciting developments in this pioneering approach are expected. This novel therapy has limitations which need to be addressed to improve efficacy, safety and clinical applicability. An overview of some of the problems and solutions is described here.
3.1 Selective targeting of oncolytic virus to tumor tissue
A major caveat for the widespread clinical use of virotherapy in canine cancer is to ensure that viruses do not harm normal cells. Certain viruses like NDV, reovirus, vesicular stomatitis virus (VSV), measles virus and others have natural tumor tropism [49–52]. However, better understanding of molecular events of virus-cell interactions in recent years has allowed for the design of genetically engineered viruses that target selected molecules or signalling pathways such as p16, p21 p53, IFN pathway, PTEN, EGFR, VEGFR, STAT3, HSP70, anti-apoptosis or hypoxia. Adenoviruses and vaccinia viruses are directed to human cancer cells by taking advantage of these defective pathways. Adenoviruses are designed to target human cancer cells mutated for tumor suppressor protein p53. The viral protein encoded by the E1 region of wild type adenovirus binds and inactivates p53, allowing replication of virus in normal cells . Because tumor cells lacking functional p53 gene are unable to suppress replication of mutant adenoviruses, E1 gene-deleted adenoviruses have diminished ability to replicate in normal cell and preserved replication in neoplastic cells . Canine p53 family proteins have biological activities similar to their human counterparts , with more than 85% gene sequence similarity. In addition, p53 mutations in canine tumors are located within the exons similar to those reported in human genes , and mutations in conserved domains of p53 appear to play a significant role in mammary carcinogenesis in both humans and dogs . Like in human tumors, the p53 gene is mutated in several canine cancers, including osteosarcoma , mammary tumors  and gastric carcinoma . All these points suggest that canine and human p53 protein has similar biological functions which can be helpful in designing better therapeutic modalities against canine cancer. Thus, oncolytic viruses, especially adenovirus, can specifically be targeted to canine cancer cells by taking advantage of the defective p53 pathway. In another example, VACV mutants with deletions in the thymidine kinase gene (tk) and/or vaccinia growth factor gene (VGF) are well advanced in pre-clinical and clinical studies for human cancers [60, 61]. These mutants grow selectively in cancer cells with high levels of cellular thymidine kinase (TK), and constitutively activated EGFR/Ras pathway signalling complements the loss of the viral gene products . Moreover, tumor cells release the TK enzyme to the circulation, probably due to disruption of dead or dying tumor cells . Increased serum TK activity was observed in various canine malignancies like lymphoma [63, 64], leukemia  and hemangiosarcoma , suggesting a possible target for directing Vaccinia virus to tumor tissue. In addition, higher expression of EGFR in mammary , glioma , hepatocellular carcinoma  and malignant epithelial nasal tumors  of canine origin closely parallels that of human tumors of the same type and histologic grade. High levels of EGFR and TK in various canine cancers could be basis of enhancing replication of Vaccinia virus in canine cancer cells.
To further enhance the tropism of vaccinia virus to human cancer cells, Kirn and colleagues deleted the B18R gene, which encodes a protein that neutralizes type I interferons (IFNs), producing a highly tumor-specific oncolytic vaccinia virus . IFNs are a group of secreted cytokines, which exert pleiotropic effects on important cell functions, including cell proliferation and modulation of the immune system [72, 73]. The IFN system also mediates the first line of cellular anti-viral response. Interestingly, about 70-75% of the cancer cells are defective in the IFN pathway . Mutation in B18R gene allows the vaccinia virus to selectively infect (cancer) cells with defects in their IFN responses but not normal cells with intact IFN responses. In this context, canine cancer cells with defects of the IFN system may be optimal targets for OVs, which exploit such defects to support their own replication.
Numerous other strategies such as transductional targeting, which use conditionally replicating viruses e.g. canine adenovirus  and transcriptional targeting, which includes use of specific promoters , may also aid in selective targeting of oncolytic viruses to cancer cells.
3.2 Spread of oncolytic viruses throughout the tumor mass
Another challenge for effective oncolytic virotherapy is the relatively poor penetration of the virus throughout solid tumor masses. As observed in human cancers, the slow spread of virus in solid tumors can be limiting and determines the outcome of therapy . The slow viral spread within solid tumors might relate to the relatively large size of OVs (e.g. around 200 nm of vaccinia virus and around 90 nm for adenovirus). Recently, Altomonte et al. showed that a single amino acid modification in the fusion protein of NDV greatly improved the fusogenicity of a recombinant virus, thereby resulting in enhanced tumor cell killing through the formation of large multi-nucleated syncytia and spread of the virus throughout the tumor mass .
The intratumoral spread and efficacy of OVs were also improved by protease or hyaluronidase mediated digestion of tumor extracellular matrix (ECM) [77–82]. Structural components of tumor ECM, such as collagens and proteoglycans, have been shown to hinder distribution of large therapeutic molecules and viruses . Therefore, the degradation of extracellular matrix with OV expressing matrix proteases and collagenases may be a useful strategy to achieve anticancer effects in both humans and dogs.
3.3 Optimization of viral replication in immune-competent hosts
Immune responses against viruses presumably limit ongoing viral replication in immunocompetent dogs. In this context, a high level of pre-existing immunity to parental viruses in canine populations might limit the use of oncolytic viruses for cancer therapy. The role of virus-neutralizing antibodies following intravenous administration remains to be determined. Use of unrelated viruses from different hosts, such as vaccinia for dog cancers, may solve the problem of pre-existing immunity. However, carrier cell based therapy also provided promising results to escape pre-existing immunity . In this regard, several types of cells, such as immune cells [84–86], stem cells [84, 87, 88] and tumor cells [89, 90] were successfully utilized as carriers of oncolytic viruses to tumors. Specific delivery to tumors and escape of the pre-existing antiviral immunity increased the effective local viral dose in the tumor tissue and thus enhanced the oncolytic effects [90–92]. However, mechanisms for the specific homing of carrier cells to tumors are currently unknown.
In a recent study, canine osteosarcoma cells treated with replication selective canine adenovirus (OCCAV) were used as carrier vehicles for evading pre-existing neutralizing antibodies against adenovirus. Systemic antitumoral activity of OCCAV, even in presence of adenovirus neutralizing antibodies, suggests a promising approach to evade pre-existing immunity against the viral vector .
The efficiency of OV replication in tumor bearing immunocompetent dogs may be enhanced by various means such as combination of viro- with chemo-  or radiation therapy  or the conjunctive use of different oncolytic viruses .
3.4 Enhancing Anti-tumor Immunity and/or Anti-tumor Effects of OVs by Virus-integrated Genes
Several strategies have been developed to achieve better anti-tumoral immunity and/or anti-tumor effects after OV cancer treatment. One strategy involves the integration of genes encoding either proteins with immunomodulatory functions, such as cytokines or chemokines, or tumor associated antigens (TAA). However, the optimal balance between anti-viral and anti-tumor immune responses is crucial for the success of these cancer therapies in immunocompetent patients.
Another strategy to enhance the OV mediated anti-cancer activity is targeting the tumor microenvironment with replicating OVs. One promising target here is the tumor neoangiogenesis. Recently, Breitbach and colleges demonstrated that VSV directly infects and destroys tumor vasculature in vivo, leaving the normal vasculature intact . In addition, armed oncolytic viruses can also prevent neoangiogenesis, leading to cancer cell necrosis. Vascular endothelial growth factor (VEGF) is a protein that plays a key role in tumor angiogenesis . Vaccinia virus encoding anti-VEGFR-1 protein decreases neoangiogenesis at the tumor site and inhibits the tumor growth . Furthermore, blocking VEGF has shown enhanced antitumor activity in a human xenograft model where vaccinia virus was armed with anti-VEGF antibody . As seen for human tumors, significant expression of VEGF enhanced angiogenesis in canine mammary gland tumors . Also, increased levels of VEGF-2 were observed in canine intracranial meningiomas , mammary adenocarcinoma , mastocytoma , mast cell tumors , and soft tissue sarcoma . Expression of VEGF in a variety of canine cancers proves its role in canine tumor angiogenesis. Thus, oncolytic viruses armed with anti-VEGF agents will be a possible therapeutic approach for canine cancers.
Finally, the anticancer activity of OVs may also be improved by expression of prodrug-converting enzymes capable of producing a toxic product within tumor tissue. Recombinant oncolytic viruses have been used to express suicide genes that convert a pro-drug into toxic drug within the tumor. Nitro-reductase enzyme from E. coli causes reduction of inactive prodrug CB1954 to promote cell killing in feline cancer cells . Similarly, 5-Fluorouracil is a pyrimidine analog widely used in canine cancer chemotherapy. Bacterial and/or yeast cytosine deaminase (CDase) is a well characterized enzyme-prodrug system that converts 5-Fluorocytosine to 5-Fluorouracil which further leads to cell cycle arrest and apoptosis leading to improved antitumor effects . Therefore, OVs armed with nitroreductase or cytosine deaminase gene in combination with prodrugs may synergistically destroy canine cancer cells.