Quantitative RT-PCR for titration of replication-defective recombinant Semliki Forest virus.
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 SFV 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.
Laboratório de Imunologia Viral, Instituto Butantan, Av. Vital Brasil 1500, CP 05503-900 São Paulo, Brazil. Electronic address: ana.puglia@butantan.gov.br.
Ana L P
AL
Puglia
Alexandre G
AG
Rezende
Soraia A C
SA
Jorge
Renaud
R
Wagner
Carlos A
CA
Pereira
Renato M
RM
Astray
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003673:Defective Viruses / Q000235:genetics / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D015203:Reproducibility of Results; D012672:Semliki forest virus / Q000235:genetics / Q000302:isolation & purification*; D019562:Viral Load / Q000379:methods*; D017361:Viral Nonstructural Proteins / Q000235:genetics; D014776:Virus Cultivation
D017361:Viral Nonstructural Proteins
Semliki Forest virus; Virus titration; qRT-PCR