| Abstract [eng] |
The prevalence of segmented RNA viruses in a variety of organisms became possible because of sequencing and bioinformatics breakthroughs. Consequently, viruses of Thogotovirus and Quaranjavirus, lesser-known genera of the Orthomyxoviridae family, were more frequently observed not only in invertebrates but also in vertebrates. These observations have sparked numerous studies focused on the zoonotic potential of new orthomyxoviruses iki cause an epidemic. Our research object is the surface glycoprotein of a quaranjavirus-like Wuhan mosquito virus 6 (WuMV-6), which is detectable in mosquito metagenomics data worldwide. The goal of this study is iki experimentally test the membrane fusion capabilities of WuMV-6 glycoprotein gp64, the sequence of which was determined using bioinformatics tools. Frist viral infection’s step - cell entry - can be tested by producing pseudotyped lentiviral vectors with WuMV-6 gp64 integrated into their envelope and infecting cells with them in vitro. Successful WuMV-6 infection can only occur if gp64 binds with cell surface receptors. We used pseudoviruses, each bearing one of three WuMV-6 gp64 sequence variants, SINUV gp64, AcMNPV gp64, or VSV G. Synthesis of C terminal hemagglutinin-tagged viral glycoproteins in mammalian HEK293T/17 cells was confirmed, although the efficiency of each glycoprotein’s synthesis differed. Pseudoviruses coated with previously mentioned glycoproteins and carrying RNA with reporter gene nanoluciferase (nLuc) were produced by HEK293T. Additionally, VSV pseudoviruses with EGFP gene transcripts were synthesized, during which the nLuc protein was also synthesized. SINUV and WuMV-6 pseudoviruses were coated with their respective gp64 but SINUV gp64 was incorporated more than WuMV-6 gp64. The quantification of pseudoviruses was determined with TaqMan qPCR. Culex tarsalis mosquito (natural WuMV-6 host) cells and chicken mononuclear blood cells were infected with pseudoviruses. Infection efficiency was evaluated by the strength of chemiluminescence from infected cells. According iki nLuc analysis, mosquito and chicken blood cells were infected with SINUV pseudoviruses. However, variation in the infection capability of pseudoviruses produced at separate times was noticed, highlighting the importance of quality checks. Nonetheless WuMV-6 gp64 successfully mediated pseudovirus entry into Cx. tarsalis cells in vitro. Cells infected with VSV pseudoviruses carrying EGFP RNA still generated a chemiluminescence signal. Due iki nLuc background, these results should be tested by another method, such as producing antibiotic resistance gene RNA transporting pseudoviruses. |
