RESUMO
Enterovirus 71 (EV71) is a causative agent of hand, foot and, mouth disease (HFMD) in young children. It is valuable for virologists to develop a fast method to rescue infectious virus from a viral cDNA clone. Here, we report a method for rapid rescue of infectious EV71 by using cells expressing T7 polymerase. The full-length EV71 genome was amplified in one step by long-distance PCR with a T7 promoter at the 5' end, and the T7 polymerase gene was cloned into a lentivirus vector for construction of a stable cell line expressing T7 polymerase. The infectious virus was rapidly and efficiently rescued by single transfection of cells with the infectious cDNA clone. Further experiments showed that the rescued virus had characteristics similar to those of the parental virus. This method circumvented the difficulty in performing in vitro transcription of a long linear DNA to obtain high-quality RNA. The construction of the viral cDNA clone and the fast rescue of the infectious virus will greatly benefit future investigations.
Assuntos
Clonagem Molecular/métodos , DNA Complementar/genética , RNA Polimerases Dirigidas por DNA/genética , Enterovirus Humano A/genética , Transfecção/métodos , Proteínas Virais/genética , Viroses/genética , Linhagem Celular , Genoma Viral/genética , Humanos , Lentivirus/genética , Reação em Cadeia da Polimerase/métodos , Regiões Promotoras Genéticas/genética , RNA Viral/genéticaRESUMO
Enterovirus 71 (EV71) causes hand-foot-and-mouth disease and severe neural complications in infants and young children. Viral pathogenesis is associated with virus-induced cell death and inflammatory cytokine production, which is usually correlated with the type of programmed cell death. EV71-infected cells were analyzed through microscopy, cell staining, and immunoblotting to determine the characteristics of EV71-induced cell death. Results demonstrated that EV71 infection induced cell shrinkage, nuclear condensation, decreased mitochondrial potential, and membrane phosphatidylserine translocation. Caspase-9 activation, poly (ADP-ribose) polymerase cleavage, and lactate dehydrogenase release were also observed during virus-induced cell death. The activated gasdermin D (GSDMD) and the phosphorylated mixed lineage kinase domain-like protein (p-MLKL) were not detected. These observations indicated that EV71-induced cell death was mainly executed by apoptosis through the intrinsic pathway rather than by GSDMD-mediated pyroptosis and p-MLKL-mediated necroptosis. Genome scanning analysis identified that EV71 2A, 2B, and 3C might be the determinant genes of virus-induced cell death. Further experiments showed that EV71 2A- and 3C-induced cell death exhibited dependence on their protease activities but involved different mechanisms. EV71 2A-induced cell death was correlated with the shut-off of host cap-dependent translation, whereas EV71 3C-induced cell death might not be ascribed to this mechanism. These findings would enhance our understanding of EV71 infection and viral pathogenesis, and help identify antiviral targets.