title: Generation of porcine reproductive and respiratory syndrome virus by in vitro assembly of viral genomic cDNA fragments.
abstract: Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent for a swine disease affecting the pig industry worldwide. Infection with PRRSV leads to reproductive complications, respiratory illness, and weak immunity to secondary infections. To better control PRRSV infection, novel approaches for generating control measures are critically needed. Here, in vitro Gibson assembly (GA) of viral genomic cDNA fragments was tested for its use as a quick and simple method to recover infectious PRRSV in cell culture. GA involves the activities of T5-exonuclease, Phusion polymerase, and Taq ligase to join overlapping cDNA fragments in an isothermal condition. Four overlapping cDNA fragments covering the entire PRRSV genome and one vector fragment were used to create a plasmid capable of expressing the PRRSV genome. The assembled product was used to transfect a co-culture of 293T and MARC-145 cells. Supernatants from the transfected cells were then passaged onto MARC-145 cells to rescue infectious virus particles. Verification and characterization of the recovered virus confirmed that the GA protocol generated infectious PRRSV that had similar characteristics to the parental virus. This approach was then tested for the generation of a chimeric virus. By replacing one of the four genomic fragments with that of another virus strain, a chimeric virus was successfully recovered via GA. In conclusion, this study describes for the first time the use of GA as a simple, yet powerful tool for generating infectious PRRSV needed for studying PRRSV biology and developing novel vaccines.
authors.affiliation: Faculty of Biotechnology, Atma Jaya Catholic University, Jl Jend, Sudirman 51, Jakarta 12930, Indonesia.
authors.forename: Maman
authors.initials: M
authors.lastname: Suhardiman
authors.affiliation: Virology and Cell Technology Laboratory, National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand Science Park, Pathumthani 12120, Thailand.
authors.forename: Jarin
authors.initials: J
authors.lastname: Kramyu
authors.affiliation: Virology and Cell Technology Laboratory, National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand Science Park, Pathumthani 12120, Thailand.
authors.forename: Jaraspim
authors.initials: J
authors.lastname: Narkpuk
authors.affiliation: Virology and Cell Technology Laboratory, National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand Science Park, Pathumthani 12120, Thailand.
authors.forename: Anan
authors.initials: A
authors.lastname: Jongkaewwattana
authors.affiliation: Virology and Cell Technology Laboratory, National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand Science Park, Pathumthani 12120, Thailand. Electronic address: nanchaya.wan@biotec.or.th.
authors.forename: Nanchaya
authors.initials: N
authors.lastname: Wanasen
mesh_terms: D000818:Animals; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D016679:Genome, Viral; D010957:Plasmids; D019316:Porcine respiratory and reproductive syndrome virus / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics*; D059386:Reverse Genetics / Q000379:methods*; D013552:Swine; D014162:Transfection
chemical_list: D018076:DNA, Complementary; D012367:RNA, Viral
keywords: Chimeric virus; Gibson assembly; MARC-145; PRRSV