Establishment of a porcine CD151 transgenic PK-15 cell line susceptible to porcine reproductive and respiratory syndrome virus

Abstract:

Abstract:［Objective］In order to study the role of porcine CD151 in infection of porcine cells by porcine reproductive and respiratory syndrome virus (PRRSV)，we established a porcine CD151 transgenic PK-15 cell line．［Methods］The full-length complementary DNA (cDNA) for porcine CD151 was amplified from porcine alveolar macrophages by reverse transcription-polymerase chain reaction (RT-PCR)，and subcloned into eukaryotic expression vector pcDNA3．The recombinant vector pcDNA-CD151 was transfected into PK-15 cells and the transgenic cell line was generated after G418 selection．Transcription of the CD151 cDNA in transgenic cell line was detected by RT-PCR and immunofluorescence．The cell line，together with control cell lines PK-15，3D4-CD163 and MARC-145，was infected with PRRSV，and the viral RNA genome or antigens in the infected cells was detected by RT-PCR or immunofluorescence．At different time points post-infection，the virus was harvested and titrated on MARC-145 cells． ［Results］The expected size of porcine CD151 cDNA was amplified with a sequence identity of 99.7% to the published sequence．From the pcDNA-CD151-transfected cell culture，a transgenic cell line PK15-CD151 was generated and correct expression of porcine CD151 was confirmed． After PRRSV infection，the viral RNA genome and antigens were detected in the cell line． Although apparent cytopathic effect was not observed in the virally infected cell line，the infectious virus with a high viral title was detected．The cell line had been passed for more than 30 generations and no significant difference in viral title was observed among generations 10，20 and 30 after PRRSV infection． ［Conclusion］Transfection of non-permissive PK-15 cells with porcine CD151 cDNA conferred the susceptibility to PRRSV infection，indicating an important role of the porcine CD151 in PRRSV infection of porcine cells．