1 Figure 1: Growth kinetics of Vero cells at different conditions.The cell culture status affects rescue of EV71 from in vitro RNA transcripts. Natallia Lazouskaya, Ruchira Makwana, Enzo A. Palombo and P. A. Barton. Environment and Biotechnology Centre, Swinburne University of Technology, Hawthorn VIC 3122 Australia Introduction Results Figure 4: Relative quantitation of EV71 RNA after transfection of Vero cells with in vitro RNA transcripts. a) RNA extracted from pooled supernatant and cells: Recovery of an infectious virus from its cDNA clone or in vitro transcribed RNA facilitates studies on many aspects of viral replication and viral pathogenicity. Several positive and negative strand RNA viruses have been successfully rescued upon transfection of susceptible cell lines with the full length cDNA viral clones or viral RNA transcripts produced by T7 or SP6 polymerases in vitro [1]. Virus yield of the recovered viruses was found to be substantially lower compared to that after transfection with virion RNA [2]. Lower infectivity of cDNA viral clones or in vitro transcribed RNA can be caused by a number of factors. Incorporation of guanine residue(s) at the 5' end of the viral genome by T7(or SP6) polymerase or an additional non-viral sequence downstream of the poly(A) tract of the cloned virus was found to inhibit viral recovery in cell culture [3]. Moreover, the shortened poly(A) tail was shown to negatively affect virus replication for a number of viruses (e.g. poliovirus, hepatitis A virus) [4, 5]. Usually, viral clones are able to restore both the authentic 3' and 5' end of the genome upon transfection in cell culture and produce infectious viral particles [6, 7]. The restoration process was predicted to happen before replication and to depend on the cell cycle and availability of host factors and enzymes required for repairing the viral genome [7, 8]. Figure 1: Growth kinetics of Vero cells at different conditions. b) RNA extracted from the cells alone: 24-well plate was seeded with 2.5x105 Vero cells/well in MEM supplemented with 5% FBS. In 24 hours, the medium was replaced with OptiMEM® I Reduced Serum Medium. Cell numbers after 24 hours incubation in OptiMEM corresponds to the cell culture immediately prior to transfection with RNA transcripts (designated as 0h on the graph). Lipofectamine2000 was added at this time point and cells were retained in OptiMEM-Lipofectamine medium for 5h. After that, medium was replaced as indicated and cells were collected and counted at the indicated times. Cell replication depended on concentration of FBS and ceased after 72 hours of incubation with the exception of cells which were passaged (at 5h p.t.) and maintained in MEM supplemented with 10% FBS. Only two experimental growth conditions were studied in real-time PCR: light blue bars represent RNA isolated from cells maintained in OptiMEM- RNA-Lipofectamine mixture for the entire period of observation; dark blue bars – the RNA from cells passaged at 5h p.t. and maintained in MEM supplemented with 10% FBS. cDNA in graph (a) was diluted 100 times prior to Q-PCR. In both experiments the negative controls showed no amplification of product. Correlation coefficients were and in (a) and (b), respectively. Aim Figure 2: CPE observed in Vero cells in 72 hours after transfection with EV71 RNA transcripts. In this study, we have examined the role of the cell culture status on rescuing Enterovirus 71 (EV71) from in vitro produced RNA transcripts. Cells were maintained in OptiMEM medium and RNA-Lipofectamine mixture for whole period of observation. Methods Conclusions OptiMEM medium and RNA-Lipofectamine mixture were replaced with MEM (5% FBS) at 5h p.t. Full length EV71 genome with T7-promoter sequence upstream of the 5UTR′ was cloned into pCR®-XL-TOPO® vector (Invitrogen). The EV71 clone was linearized downstream of the poly(A) tail and used as a template in in vitro transcription reaction with RiboMAX™ Large Scale RNA Production System–T7 (Promega). The in vitro RNA transcripts were transfected into Vero cell culture in the presence of Lipofectamine™ 2000 (Invitrogen). Prior to transfection, Vero cells were seeded and grown for 24 hours in Minimum Essential Medium (MEM, Sigma) supplemented with 5% fetal bovine serum (FBS, Invitrogen). The growth medium was replaced with OptiMEM® I Reduced Serum Medium (Invitrogen) for another 24 hours. Five hours after transfection the OptiMEM medium was removed from designated wells and the cells were maintained at the following growth conditions: MEM supplemented with 5% FBS; MEM supplemented with 10% FBS; Cells were passaged 5h post transfection (p.t.) and maintained in MEM with 10% FBS; One cell culture was retained in reduced serum medium containing the RNA transcripts for the whole period of incubation (6 days). The cytopathic effect (CPE) was observed microscopically. At 144 hours p.t. the supernatant and cells were collected and viral titers were calculated in TCID50 assay using the Reed-Muench method. The TCID50 units were converted to plaque forming units (PFU) using Kärber’s formula: 1 TCID50 = 0.69 PFU Real-time PCR was preformed to monitor the changes in viral RNA concentration inside the transfected cells and in the supernatant. Microscopic observation showed that CPE developed faster in serum-depleted cell culture where the transfected RNA was left for 6 days. However, the resulting viral titers were higher in actively dividing cells despite the removal of the RNA transcripts 5 hours p.t. Q-RT-PCR analysis showed that RNA uptake by cells continued beyond the 5 hours incubation period. Nevertheless, the quiescent cells failed to produce viral titers equal to those found in actively dividing cells. The data demonstrated that growth conditions of cell culture after transfection can significantly affect recovery of the virus from in vitro transcribed RNA. OptiMEM medium and RNA-Lipofectamine mixture were replaced with MEM (10% FBS) at 5h p.t. Cells were passaged at 5h p.t. and grown in MEM (10% FBS). References Figure 3: Total number of PFU of EV71 recovered under different cell culture conditions. 1. BOYER, J. C. & HAENNI, A. L Infectious transcripts and cDNA clones of RNA viruses. Virology, 198, 2. VAN DER WERF, S., BRADLEY, J., WIMMER, E., STUDIER, F. W. & DUNN, J. J Synthesis of infectious poliovirus RNA by purified T7 RNA polymerase. Proc Natl Acad Sci U S A, 83, 3. SARNOW, P Role of 3'-end sequences in infectivity of poliovirus transcripts made in vitro. J Virol, 63, 4. SILVESTRI, L. S., PARILLA, J. M., MORASCO, B. J., OGRAM, S. A. & FLANEGAN, J. B Relationship between poliovirus negative-strand RNA synthesis and the length of the 3' poly(A) tail. Virology, 345, 5. HEROLD, J. & ANDINO, R Poliovirus RNA Replication Requires Genome Circularization through a Protein-Protein Bridge. Molecular Cell, 7, 6. KUSOV, Y. Y., GOSERT, R. & GAUSS-MULLER, V Replication and in vivo repair of the hepatitis A virus genome lacking the poly(A) tail. J Gen Virol, 86, 7 LIU, G. Q., NI, Z., YUN, T., YU, B., ZHU, J. M., HUA, J. G. & CHEN, J. P Rabbit hemorrhagic disease virus poly(A) tail is not essential for the infectivity of the virus and can be restored in vivo. Arch Virol, 153, 8. FEUER, R., MENA, I., PAGARIGAN, R., SLIFKA, M. K. & WHITTON, J. L Cell cycle status affects coxsackievirus replication, persistence, and reactivation in vitro. J Virol, 76, Total virus production after transfection Vero cells with in vitro transcribed RNA was 56.4 fold higher in actively dividing cell culture.