Human telomerase reverse transcriptase-immortalized porcine monomyeloid cell lines for the production of porcine reproductive and respiratory syndrome virus.

摘要

Porcine reproductive and respiratory syndrome virus (PRRSV) shows highly restricted cell tropism and targets subpopulations of differentiated macrophages such as porcine alveolar macrophages (PAM) in the natural host. Although primary PAM cells would be ideal for in vitro virus production, they are not only difficult and expensive for establishment but cannot be frozen reliably for long-term storage and use. Apart from PAM cells, African green monkey kidney derived Marc-145 cells are used commonly for virus propagation. However, concerns have been raised regarding a possible modification of specific epitopes associated with virus neutralization because of distinct virus entry between PAM and Marc-145 cells. In order to overcome these problems, the present study was aimed to generate immortalized porcine monocyte/macrophage cell lines and to evaluate their potential for PRRSV production. Primary PAM cells were transfected stably with the human telomerase reverse transcriptase (hTERT) cDNA by a retrovirus vector so that constitutive expression of the hTERT protein allows cells to proliferate indefinitely. The newly immortalized PAM clones were shown to exert functional telomerase activity, indicating sustained expression of hTERT. In addition, telomerase-immortalization of PAMs did not affect expression levels of the native CD163 receptor on their surface. It was further demonstrated that these continuous PAM cell lines are fully permissive for the efficient growth of both type 1 and 2 PRRSV strains. The findings suggest that the hTERT-immortalized PAM cell lines can enable us to facilitate the continued use of PAMs for virus isolation and production and to provide a promising tool for viral pathogenesis and immune function studies.