by the state agency of medicines of the republic of latvia in 2004. it was also approved in georgia and armenia. it is wild type echo-7, a member of echovirus group. the potential use of echovirus as an oncolytic virus to treat cancer was discovered by latvian scientist aina muceniece in the 1960s and 1970s. the data used to register the drug in latvia is not sufficient to obtain approval to use it in the us, europe, or japan. as of 2017 there was no good evidence that rigvir is an effective cancer treatment. on 19 march 2019, the manufacturer of echo-7, sia latima, announced the drug's removal from sale in latvia, quoting financial and strategic reasons and insufficient profitability. however, several days later an investigative tv show revealed that state agency of medicines had run laboratory tests on the vials, and found that the amount of echo-7 virus is of a much smaller amount than claimed by the manufacturer. according to agency's lab director, "it's like buying what you think is lemon juice, but finding that what you have is lemon-flavored water". in march 2019, the distribution of echo-7 in latvia has been stopped. based on the request of some patients, medical institutions and physicians were allowed to continue use despite the suspension of the registration certificate. semliki forest virus semliki forest virus (sfv) is a virus that naturally infects cells of the central nervous system and causes encephalitis. a genetically engineered form has been pre-clinically tested as an oncolytic virus against the severe brain tumour type glioblastoma. the sfv was genetically modified with microrna target sequences so that it only replicated in brain tumour cells and not in normal brain cells. the modified virus reduced tumour growth and prolonged survival of mice with brain tumours. the modified virus was also found to efficiently kill human glioblastoma tumour cell lines. other the maraba virus, first identified in brazilian sandflies, is being tested clinically. coxsackievirus a21 is being developed by viralytics under trade name cavatak. coxsackievirus a21 belongs to enterovirus c species. influenza a is one of the earliest viruses anecdotally reported to induce cancer regression. this has prompted preclinical development of genetically engineered oncolytic influenza a viruses. engineering oncolytic viruses directed evolution an innovative approach of drug development termed "directed evolution" involves the creation of new viral variants or serotypes specifically directed against tumour cells via rounds of directed selection using large populations of randomly generated recombinant precursor viruses. the increased biodiversity produced by the initial homologous recombination step provides a large random pool of viral candidates which can then be passed through a series of selection steps designed to lead towards a pre-specified outcome (e.g. higher tumor specific activity) without requiring any previous knowledge of the resultant viral mechanisms that are responsible for that outcome. the pool of resultant oncolytic viruses can then be further screened in pre-clinical models to select an oncolytic virus with the desired therapeutic characteristics. directed evolution was applied on human adenovirus, one of many viruses that are being developed as oncolytic agents, to create a highly selective and yet potent oncolytic vaccine. as a result of this process, coload1 (a novel chimeric member of the group b adenoviruses) was generated. this hybrid of adenovirus serotypes ad11p and ad3 shows much higher potency and tumour selectivity than the control viruses (including ad5, ad11p and ad3) and was confirmed to generate approximately two logs more viral progeny on freshly isolated human colon tumour tissue than on matching normal tissue. attenuation attenuation involves deleting viral genes, or gene regions, to eliminate viral functions that are expendable in tumour cells, but not in normal cells, thus making the virus safer and more tumour-specific. cancer cells and virus-infected cells have similar alterations in their cell signalling pathways, particularly those that govern progression through the cell cycle. a viral gene whose function is to alter a pathway is dispensable in cells where the pathway is defective, but not in cells where the pathway is active. the enzymes thymidine kinase and ribonucleotide reductase in cells are responsible for dna synthesis and are only expressed in cells which are actively replicating. these enzymes also exist in the genomes of certain viruses (e.g. hsv, vaccinia) and allow viral replication in quiescent(non-replicating) cells, so if they are inactivated by mutation the virus will only be able to replicate in proliferating cells, such as cancer cells. tumour targeting there are two main approaches for generating tumour selectivity: transductional and non-transductional targeting. transductional targeting involves modifying the viral coat proteins to target tumour cells while reducing entry to non-tumour cells. this approach to tumour selectivity has mainly focused on adenoviruses and hsv-1, although it is entirely viable with other viruses. non-transductional targeting involves altering the genome of the virus so it can only replicate in cancer cells, most frequently as part of the attenuation of the virus. transcription targeting can also be used, where critical parts of the viral genome are placed under the control of a tumour-specific promoter. a suitable promoter should be active in the tumour but inactive in the majority of normal tissue, particularly the liver, which is the organ that is most exposed to blood born viruses. many such promoters have been identified and studied for the treatment of a range of cancers. similarly, viral replication can be finely tuned with the use of micrornas (mirna) artificial target sites or mirna response elements (mres). differential expression of mirnas between healthy tissues and tumors permit to engineer oncolytic viruses detargeted from certain tissues of interest while allowing its replication in the tumor cells. double targeting with both transductional and non-transductional targeting methods is more effective than any one form of targeting alone. further information: oncolytic adenovirus reporter genes viral luciferase expression in a mouse tumour both in the laboratory and in the clinic it is useful to have a simple means of identifying cells infected by the experimental virus. this can be done by equipping the virus with "reporter genes" not normally present in viral genomes, which encode easily identifiable protein markers. one example of such proteins is gfp (green fluorescent protein) which, when present in infected cells, will cause a fluorescent green light to be emitted when stimulated by blue light. an advantage of this method is that it can be used on live cells and in patients with superficial infected lesions, it enables rapid non-invasive confirmation of viral infection. another example of a visual marker useful in living cells is luciferase, an enzyme from the firefly which in the presence of luciferin, emits light detectable by specialized cameras. vaccinia virus infected cells expressing beta-glucuronidase (blue colour) the e. coli enzymes beta-glucuronidase and beta-galactosidase can also be encoded by some viruses. these enzymes, in the presence of certain substrates, can produce intense colored compounds useful for visualizing infected cells and also for quantifying gene expression. modifications to improve oncolytic activity oncolytic viruses can be used against cancers in ways that are additional to lysis of infected cells. suicide genes viruses can be used as vectors for delivery of suicide genes, encoding enzymes that can metabolise a separately administered non-toxic pro-drug into a potent cytotoxin, which can diffuse to and kill neighbouring cells. one herpes simplex virus, encoding a thymidine kinase suicide gene, has progressed to phase iii clinical trials. the herpes simplex virus thymidine kinase phosphorylates the pro-drug, ganciclovir, which is then incorporated into dna, blocking dna synthesis. the tumour selectivity of oncolytic viruses ensures that the suicide genes are only expressed in cancer cells, however a "bystander effect" on surrounding tumour cells has been described with several suicide gene systems. suppression of angiogenesis angiogenesis (blood vessel formation) is an essential part of the formation of large tumour masses. angiogenesis can be inhibited by the expression of several genes, which can be delivered to cancer cells in viral vectors, resulting in suppression of angiogenesis, and oxygen starvation in the tumour. the infection of cells with viruses containing the genes for angiostatin and endostatin synthesis inhibited tumour growth in mice. enhanced antitumour activities have been demonstrated in a recombinant vaccinia virus encoding anti-angiogenic therapeutic antibody and with an hsv1716 variant expressing an inhibitor of angiogenesis. radioiodine adenoviral nis gene expression in a mouse tumour (located at the crosshairs) following intravenous delivery of virus (left) compared to an uninfected control mouse (right) addition of the sodium-iodide symporter (nis) gene to the viral genome causes infected tumour cells to express nis and accumulate iodine. when combined with radioiodine therapy it allows local radiotherapy of the tumour, as used to treat thyroid cancer. the radioiodine can also be used to visualise viral replication within the body by the use of a gamma camera. this approach has been used successfully preclinically with adenovirus, measles virus and vaccinia virus. approved therapeutic agents talimogene laherparepvec (oncovex gm-csf), aka t-vec, by amgen, successfully completed phase iii trials for advanced melanoma in march 2013. in october 2015, the us fda approved t-vec, with the brand name imlygic, for the treatment of melanoma in patients with inoperable tumors. becoming the first approved oncolytic agent in the western world. it is based on herpes simplex virus (hsv-1). it has also been tested in a phase i trial for pancreatic cancer and a phase iii trial in head and neck cancer together with cisplatin chemotherapy and radiotherapy. teserpaturev (g47∆), aka delytact by daiichi sankyo is a first oncolytic virus therapy approved by japan ministry of health, labour and welfare (mhlw). delytact is a genetically engineered oncolytic herpes simplex virus type 1 (hsv-1) approved for treatment of malignant glioma in japan. oncolytic viruses in conjunction with existing cancer therapies it is in conjunction with conventional cancer therapies that oncolytic viruses have often showed the most promise, since combined therapies operate synergistically with no apparent negative effects. clinical trials onyx-015 (dl1520) underwent trials in conjunction with chemotherapy before it was abandoned in the early 2000s. the combined treatment gave a greater response than either treatment alone, but the results were not entirely conclusive. vaccinia virus gl-onc1 was studied in a trial combined with chemo- and radiotherapy as standard of care for patients newly diagnosed with head & neck cancer. herpes simplex virus, adenovirus, reovirus and murine leukemia virus are also undergoing clinical trials as a part of combination therapies. pre-clinical research chen et al. (2001) used cv706, a prostate-specific adenovirus, in conjunction with radiotherapy on prostate cancer in mice. the combined treatment resulted in a synergistic increase in cell death, as well as a significant increase in viral burst size (the number of virus particles released from each cell lysis). no alteration in viral specificity was observed. seprehvir (hsv-1716) has also shown synergy in pre-clinical research when used in combination with several cancer chemotherapies. the anti-angiogenesis drug bevacizumab (anti-vegf antibody) has been shown to reduce the inflammatory response to oncolytic hsv and improve virotherapy in mice. a modified oncolytic vaccinia virus encoding a single-chain anti-vegf antibody (mimicking bevacizumab) was shown to have significantly enhanced antitumor activities than parental virus in animal models. in fiction in science fiction, the concept of an oncolytic virus was first introduced to the public in jack williamson's novel dragon's island, published in 1951, although williamson's imaginary virus was based on a bacteriophage rather than a mammalian virus. dragon's island is also known for being the source of the term "genetic engineering". the plot of the hollywood film i am legend is based on the premise that a worldwide epidemic was caused by a viral cure for cancer. in the fox broadcasting company tv series house season 2 episode 19, a strain of herpes simplex virus is shown to have shrunk a hepatic tumor.