Glioblastoma multiforme is the most lethal type of brain tumor that is not yet curable owing to its frequent resurgence after surgery. Resistance is mainly caused by the presence of a subpopulation of tumor cells, the glioma stem cells (GSCs), which are highly resistant to radiation and chemotherapy. In 2015, Zikavirus (ZIKV)-induced microcephaly emerged in newborns, indicating that ZIKV has a specific neurotropism. Accordingly, an oncolytic tropism for infecting GSCs was demonstrated in a murine tumor model. Like other flaviviruses, ZIKV is enveloped by two proteins, prM and E. The pME expression plasmid along with the HIV-1 vector pNL Luc AM generated prME pseudotyped viral particles. Four different prME envelopes, Z1 to Z4, were cloned, and the corresponding pseudotypes, Z1-to Z4-HIVluc, produced by this two-plasmid system, were tested for entry efficiency using Vero-B4 cells. The most efficient pseudotype, Z1-HIVluc, also infected glioma-derived cell lines U87 and 86HG39. The pseudotype system was then extended by using a three-plasmid system including pME-Z1, the HIV-1 packaging plasmid psPAX2, and the lentiviral vector pLenti-luciferase-P2A-Neo. The corresponding pseudotype, designated Z1-LENTIluc, also infected U87 and 86HG39 cells. Altogether, a pseudotyped virus especially targeting glioma-derived cells might be a promising candidate for a prospective glioblastoma-directed virotherapy. Cancers 2020, 12, 1000 2 of 18 biology, leading to the generation of recombinant, genetically modified virus candidates. Clinical trials have been completed using modified versions of herpes simplex virus [7-12], adenovirus [13,14], Newcastle disease virus [15], reovirus [16,17], parvovirus [18], and poliovirus [19]. Some of these studies demonstrated that a small subset of patients had a benefit from such virotherapy applications, but the statistical significance of these findings must be demonstrated in larger control trials [20].A new discovery, with a high impact on the future aspects of glioma virotherapy, was that Zikavirus (ZIKV) showed a specific neurotropism for glial cells [21]. ZIKV is a new emerging mosquito-borne virus belonging to the Flaviviridae family [22]. In the Flaviviridae family, all members are enveloped viruses with two external, membranous proteins, the envelope E and the precursor of the membrane protein prM [23], both relevant for viral entry. In early 2015, abnormal rates of ZIKV infections were seen in Brazil, and in late 2015, an unexpected high number of newborns showed microcephaly, a disease where a baby's head is much smaller than expected. Altogether, the new emerging ZIKV was shown to be the cause of Guillain-Barré syndrome (GBS), microcephaly, and other congenital brain abnormalities, proving its neurotropic phenotype [24]. An interesting finding was the oncolytic activity of ZIKV against GSCs [25]. A selective effect of ZIKV was observed for GSCs, causing loss of self-renewal and proliferation in glioblastoma organoids. In mice, ZIKV infection prolonged survival by slowing down tumor g...