| Full text | |
| Author(s): |
Puglia, Ana L. P.
[1]
;
Rezende, Alexandre G.
[1]
;
Jorge, Soraia A. C.
[1]
;
Wagner, Renaud
[2]
;
Pereira, Carlos A.
[1]
;
Astray, Renato M.
[1]
Total Authors: 6
|
| Affiliation: | [1] Inst Butantan, Lab Imunol Viral, BR-05503900 Sao Paulo - Brazil
[2] Univ Strasbourg, CNRS, Ecole Biotechnol Strasbourg, Inst Rech, F-67412 Illkirch Graffenstaden - France
Total Affiliations: 2
|
| Document type: | Journal article |
| Source: | Journal of Virological Methods; v. 193, n. 2, p. 647-652, NOV 2013. |
| Web of Science Citations: | 7 |
| Abstract | |
Virus titration may constitute a drawback in the development and use of replication-defective viral vectors like Semliki Forest virus (SFV). The standardization and validation of a reverse transcription quantitative PCR (qRT-PCR) method for SFV titration is presented here. The qRT-PCR target is located within the nsp1 gene of the non-structural polyprotein SFV region (SFV RNA), which allows the strategy to be used for several different recombinant SFV constructs. Titer determinations were carried out by performing virus titration and infection assays with SFVs containing an RNA coding region for the rabies virus glycoprotein (RVGP) or green fluorescent protein (GFP). Results showed that the standardized qRT-PCR is applicable for different SFV constructs, and showed good reproducibility. To evaluate the correlation between the amount of functional SFV RNA in a virus lot and its infectivity in BHK-21 cell cultures, a temperature mediated titer decrease was performed and successfully quantitated by qRT-PCR. When used for cell infection at the same multiplicity of infection (MOI), the temperature treated SFV-RVGP samples induced the same levels of RVGP expression. Similarly, when different SFV-GFP lots with different virus titers, as accessed by qRT-PCR, were used for cell infection at the same MOI, the cultures showed comparable amounts of fluorescent cells. The data demonstrate a good correlation between the amount of virus used for infection, as measured by its SW RNA, and the protein synthesis in the cells. In conclusion, the qRT-PCR method developed here is accurate and enables the titration of replication-defective SFV vectors, an essential aid for viral vector development as well as for establishment of production bioprocesses. (C) 2013 Elsevier B.V. All rights reserved. (AU) | |
| FAPESP's process: | 11/15269-0 - Construction and utilization of SFV vector expressing GFP and RVGP |
| Grantee: | Ana Lia Pradella Puglia |
| Support Opportunities: | Scholarships in Brazil - Master |
| FAPESP's process: | 10/08742-8 - Evaluation of immune response in mice immunized with Semliki Forest Virus (SFV) carrying the genetic information for in vivo synthesis of rabies virus glycoprotein (RVGP) |
| Grantee: | Renato Mancini Astray |
| Support Opportunities: | Regular Research Grants |