Tumor cells have specific and high surface expression of the oncolytic viral primary receptor, which is capable of binding to viral surface proteins via the receptor-ligand pathway, essential for transduction. The first step is absorption, followed by the viral surface adhesion protein combining with the target cell surface receptor, which initiates endocytic signaling pathways (13). A number of viruses have a natural tropism for tumor cells. This principle is illustrated by the susceptibility of malignant glioma cells to poliovirus, depending on the presence of CD155 on glial neoplasms (14,15). Alternatively, in order to infect the targeted tumor cells, the viral surface binding protein, which has the ability to serve as a receptor, must transform by introducing a single chain antibody or a polypeptide binding ligand: For example, the urokinase receptor (uPAR) is overexpressed in multiple malignancies and serves a role in tumor invasion and angiogenesis (16,17). Measles virus retargeted against human (MV-h-uPA) or mouse (MV-m-uPA) uPAR were constructed by superinducing the aminoterminal fragment of either human or mouse urokinase in the C-terminus of recombinant measles virus. In vitro experiments indicated that MV-h-uPA and MV-m-uPA were able to specifically infect cancer cells that overexpressed uPAR via the receptor-ligand pathway (18).

The approach to achieving tumor-selective viral replication has been to alter the function of the control of genetic transcription, which is essential in viral replication, to a tissue- or tumor-specific promoter. These promoters include human telomerase reverse transcription (hTERT) (19), hypoxia-inducible factor-1 (20), prostate-specific antigen (21) and α-fetoprotein (22), among others. hTERT has been identified as a major protein that functions to maintain telomere length in tumors; however, it demonstrates little or no expression in normal cells, allowing cancer cells to subvert the Hayflick limit (23). An attenuated adenovirus 5 vector, OBP-301, was constructed, whereby the hTERT promoter element drives the expression of E1 genes: Due to only tumor cells with telomerase activity possessing the ability to activate this promoter, selective viral replication and oncolytic cell death was demonstrated (19).

In addition to this approach, a novel system, which may regulate oncolytic viral gene-targeted expression by exploiting microRNAs (miRNAs), has been developed. miRNAs are 20–22 nucleotides of small noncoding endogenously produced RNAs that can base pair to their target mRNAs. This enables them to guide post-transcriptional silencing of their target genes and determine the differential expression between cancerous and normal tissues (24). The negative regulation in gene expression makes it possible to utilize these miRNAs to inhibit viral replication in normal cells (25,26). Aberrant expression of miRNAs has previously been observed in numerous types of cancer (27). The downregulation of miRNA (miR)-199 occurs consistently in almost all hepatocellular carcinomas (HCC), when compared with a normal liver (28). Additionally, a conditionally replication-competent oncolytic adenovirus (OAd), ad-199T, was generated by introducing miR-199 target sites within the 3′untranslated region of the E1A gene, essential for viral replication, in order that miR-199 demonstrated the capacity to negatively regulate E1A gene expression. As a result, ad-199T replication was inhibited in normal miR-199-positive liver parenchyma, and unaffected in tumor cells with low expression of miR-199 (29).

Type I interferons (IFNs), which are spontaneously produced in response to a viral infection, are an important cytokine (12). The mechanism of cancer evolution that causes the loss of antiviral responsiveness in the majority of cancer cell lines, particularly the activity of IFN-regulated signaling pathways, is not completely understood. Previous studies have revealed that tumor antiviral activity is incompatible with their own efficient cell growth, as IFN and IFN-responsive genes are known angiogenesis inhibitors (30), and are also recognized for their capacity to induce apoptosis (31). In addition, the deficiency of tumor antiviral activity renders it more susceptible to an infection compared with normal cells, which results in a survival advantage for viruses within tumor cells. The vesicular stomatitis virus (VSV) is sensitive to IFN and therefore has an advantage in cancer therapeutics, including using a defective IFN pathway, to preferentially infect and kill tumor cells (32). The targeting of cancer cells by the VSV can be improved by introducing mutations in the matrix (M) protein. The M protein of VSV is a major structural protein that functions in virus assembly, as well as in the suppression of host gene expression, causing inhibition of IFNs and other antiviral proteins. The ability of the M protein to subdue host gene expression is genetically distinct from its viral assembly function. Furthermore, various mutations render the M protein defective in its ability to suppress host gene expression, without subverting its ability to function in viral assembly (33). As a result, normal cells, due to the activated IFN pathway, inhibit the M protein mutant VSV replication; otherwise cancer cells with defects in the IFN pathway are killed by M protein mutant VSV selective replication (34).

TP53 serves a key function in inducing apoptosis by prompting cell cycle arrest, due to activation in response to oncogene activation, DNA damage and other stress signals (35). p53 is the most frequently mutated gene in cancer, being altered in >50% of all types of human malignancies (36). The mutated p53 gene is an important hallmark of cancer (37). Viruses have evolved the ability to inhibit the apoptosis of host cells, in order to accomplish viral replication. The E1B55K adenovirus codes for the protein that inactivates the p53 protein of host cells, contributing to self-proliferation and virus replication (38). China approved the world's first OV therapy for cancer treatment (39). OV, also termed H101, has been approved for the treatment of numerous types of cancer clinically (40), H101 is a type of OAd with E1B-55KD and partial E3 deletion, and it cannot replicate in normal cells where p53 is active; therefore, H101 can selectively infect and kill tumor cells via the targeting of pro-apoptosis (41,42).

An important reason why OVs are currently being advanced as a promising antitumor modality is that they are able to transfer and amplify therapeutic genes, whilst simultaneously avoiding immunosurveillance by infected host cells (43). Virosomes are utilized as vectors for the delivery of toxic genes, including immunostimulatory genes [for instance, interleukin-12 (IL-12) (44)], anti-angiogenic genes (45), prodrug-converting enzyme genes and pro-apoptotic genes, to advance its potency, which can be used for oncotherapy (46).

Delivery of immunostimulatory genes to cancer cells should boost immune responses against tumor antigens and the activity of cytotoxic effector cells, as a result of an abnormal increase of inflammatory infiltrates in the tumor milieu (47). Currently, a variety of immunostimulatory genes are used as part of recombinant oncolytic virusal vectors, which are utilized in viral gene therapy, including granulocyte-macrophage colony stimulating factor (GM-CSF) (48), IL-2, IL-12, IL-15 and IL-18 (49,50), IFN-α and IFN-β (51,52). Previously, Choi et al (49) generated IL-12- and IL-18-expressing (Ad-ΔE1Bmt7/IL-12/IL-18) OAd. IL-12 has been demonstrated to directly activate cytotoxic T cells and natural killer (NK) cells, which then produce high levels of IFN-γ, enhance their cytolytic activity and induce an antitumor effect (46); however, despite encouraging results in animal models, weaker antitumor effects of IL-12 were recorded in early clinical trials, and frequently accompanied by unacceptable levels of adverse events (53,54). This markedly dampened the hopes of the clinical application of this cytokine in patients with cancer. In order to minimize the adverse events of an IL-12-based therapy, a decrease in the systemic expression of IL-12 has been the selected approach. This approach involves patients who are injected intratumorally with Ad-RTS-hIL-12, an adenoviral vector engineered for the controlled expression of IL-12. Preliminary results were encouraging, with positive clinical efficacy observed in 5/7 patients treated with Ad-RTS-IL-12. These responses were associated with intratumoral IL-12 mRNA expression, reflected as a decrease in the size of the injected and distant lesions (55). IL-18 serves a function in inducing the differentiation of antitumor effector T cells and the activation of NK cells, which exhibit potent cytotoxicity against tumor cells (56). In a B16-F10 murine melanoma model, IL-12, IL-18, IFN-γ and GM-CSF levels within the tumor tissues were significantly increased when treated with the intratumoral application of OAd co-expressing IL-12 and IL-18 compared with control virus-treated mice. Higher numbers of CD4⁺ T cells, CD8⁺ T cells and NK cells were detected via an immunohistochemical staining and the histological evaluation of tumor sections, which also exhibited large areas of necrosis compared with control virus-treated mice. These results further demonstrated the enhanced antitumor effects of OAd that express IL-12 and IL-18. The results were also validated in a murine colon adenocarcinoma model of MC38cea (48). IL-12- and IL-18-based immunotherapies were particularly efficacious in patients with cancer that contained inherited defects in IL-12 or IL-18 production, or with downregulated expression of IL-12 or IL-18. Compared with control virus-treated mice, the intratumoral administration of GM-CSF-expressing oncolytic MV markedly prolonged the median overall survival time (49).

OVs were engineered to express prodrug-converting enzymes, which caused the conversion of the non-toxic prodrug into a toxic form, at the site of viral replication; therefore, causing selective tumor cell death. The prodrug-converting enzymes applied to the oncolytic virotherapy, including thymidine kinase (TK) (57), cytosine deaminase (CD) and purine-nucleoside phosphorylase (PNP) (58–60). The mechanism of action involves the conversion of the prodrug ganciclovir (GCV), via TK, into ganciclovir triphosphate (GCV-TP), CD converts the prodrug 5-fluorocytosine (5-FC) into 5-fluorouracil (5-FU) and PNP converts the prodrug fludarabine phosphate into 2-fluoroadenine. The transformed products are nucleoside analogs, which interfere with DNA replication in actively dividing tumor cells via the inhibition of DNA synthesis (61). A new recombinant herpes simplex virus type 1 thymidine kinase (HSV-1-TK), containing BoHV-4, has been constructed by Redaelli et al (62). The HSV-TK enzyme, which is an enzyme homologous to that of TK, has 1,000 times more affinity for the substrate GCV than the host cell TK (63). HSV-1-TK in combination with GCV has been identified as a promising suicide gene system, whereby GCV is phosphorylated by HSV-1-TK to form GCV-TP. GCV-TP has the ability to inhibit DNA polymerases, resulting from competing with deoxyguanosine triphosphate to bind to DNA polymerases, causing DNA damage that leads to cell death (62,64). The active metabolite of TK, PNP and 5-FU can directly kill infected tumor cells. Furthermore, they promote a strong bystander effect on neighboring tumor cells that are not actively infected (59,65,66). In a previous study by Leveille et al (67), recombinant vesicular stomatitis virus (VSV-MD51) expressing the cytosine deaminase/uracil phosphoribosyl-transferase suicide gene with the 5-FC prodrug was combined for the treatment of cancer. It was observed that there was a synergistic effect on killing cancer cells with VSV-MD51 and 5-FU in several cancer cell lines. The high solubility of 5-FU is lethal to non-infected bystander tumor cells, which is an important advantage in using this combination.

In 1971, Folkman (68), to the best of our knowledge, first produced the hypothesis that tumor growth depends on angiogenesis, which also serves an essential role in tumor invasion and metastasis. Within four decades, anti-angiogenic therapy with broad targeting has rapidly evolved to become an integral component of current standard anticancer treatments (69,70). Endostatin and angiostatin are two endogenous and broad-spectrum angiogenesis inhibitors. These inhibitors only function when they are continuously transported to the tumor microenvironment (71,72). Additionally, it is difficult to make the two endogenous inhibitors function in the traditional way, due to characteristics of short serum half-lives, low solubility and poor stability (73,74). Hutzen et al (73) developed recombinant MVs that express human and mouse variants of endostatin: Angiostatin (E:A) fusion proteins, known as MV-hE:A and MV-mE:A, respectively. The in vitro study revealed that there is active MV replication and concomitant continuous expression of target genes within the medulloblastoma cells; therefore, angiogenic factors were inhibited, leading to a significant decrease in endothelial cell growth, viability and migration (73).