Reverse genetic manipulation of the overlapping coding regions for structural proteins of the type II porcine reproductive and respiratory syndrome virus.
The overlapping genomic regions coding for structural proteins of porcine reproductive and respiratory syndrome virus (PRRSV) poses problems for molecular dissection of the virus replication process. We constructed five mutant full-length cDNA clones with the overlapping regions unwound and 1 to 3 restriction sites inserted between two adjacent ORFs (ORF1/2, ORF4/5, ORF5/6, ORF 6/7 and ORF7/3' UTR), which generated the recombinant viruses. Our findings demonstrated that 1) the overlapping structural protein ORFs can be physically separated, and is dispensable for virus viability; 2) such ORF separations did not interrupt the subgenomic RNA synthesis; 3) the plaque morphology, growth kinetics, and antigenicity of these mutant viruses were virtually indistinguishable from those of the parental virus in cultured cells; and 4) these mutant viruses remained genetic stable in vitro. This study lays a foundation for further molecular dissection of PRRSV replication process, and development of genetically tagged vaccines against PRRS.
Yu
Dandan
D
Department of Swine Infectious Diseases, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology, Chinese Ministry of Agriculture, Shanghai 200241, China.
Lv
Jian
J
Sun
Zhi
Z
Zheng
Haihong
H
Lu
Jiaqi
J
Yuan
Shishan
S
D000956:Antigens, Viral / Q000276:immunology; D016679:Genome, Viral; D050296:Microbial Viability; D008958:Models, Molecular; D008967:Molecular Biology / Q000379:methods; D009690:Nucleic Acid Conformation; D019316:Porcine respiratory and reproductive syndrome virus / Q000235:genetics* / Q000502:physiology; D012367:RNA, Viral / Q000096:biosynthesis / Q000235:genetics; D011995:Recombination, Genetic; D010948:Viral Plaque Assay; D015678:Viral Structural Proteins / Q000235:genetics*; D014779:Virus Replication
D000956:Antigens, Viral; D012367:RNA, Viral; D015678:Viral Structural Proteins