Detection of replication competent adenovirus upon serial passaging of recombinant adenovirus expressing FMDV capsid proteins.
Replication deficient human adenovirus type 5 (hAd5) is an important gene delivery vehicle and has been used in various fields of biomedical sciences such as gene therapy, cancer therapy and vaccination. Inspite of its various useful features, emergence of replication competent adenovirus (RCA) in recombinant virus stocks is a great concern. In the present study, recombinant adenovirus expressing foot-and-mouth disease virus serotype-O capsid proteins was propagated in HEK-293 cells and purified by CsCl density gradient ultracentrifugation. The virus was serially passaged in HEK-293 cells and at passage level (P) 2-4, 6, 8 and 12, tested for the presence of RCA. A vector dose of 2 × 10(8) and 1 × 10(10) TCID50 of the virus was used in cell line bioassay and PCR, respectively. Our results demonstrated that the PCR is more sensitive and rapid technique for the detection of RCA in recombinant adenovirus stocks as early as at P3, whereas the bioassay detected the RCA at P8.
Indian Veterinary Research Institute, Hebbal Campus, Bangalore 560 024, India.
Ramesh
R
Kumar
Indian Veterinary Research Institute, Hebbal Campus, Bangalore 560 024, India.
Suresh H
SH
Basagoudanavar
Indian Veterinary Research Institute, Hebbal Campus, Bangalore 560 024, India. Electronic address: bpsrini@gmail.com.
B P
BP
Sreenivasa
D000256:Adenoviridae; D036022:Capsid Proteins / Q000096:biosynthesis / Q000235:genetics; D029544:Foot-and-Mouth Disease Virus / Q000235:genetics*; D005822:Genetic Vectors; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D011994:Recombinant Proteins; D014161:Transduction, Genetic; D014779:Virus Replication
D036022:Capsid Proteins; D011994:Recombinant Proteins
Cell line bioassay; FMD virus; HEK-293; Human adenovirus type 5; PCR; Replication competent adenovirus