Susceptible cell lines for the production of porcine reproductive and respiratory syndrome virus by stable transfection of sialoadhesin and CD163

Abstract

Background: Porcine reproductive and respiratory syndrome virus (PRRSV) causes major economic losses in the pig industry worldwide. In vivo, the virus infects a subpopulation of tissue macrophages. In vitro, PRRSV only replicates in primary pig macrophages and African green monkey kidney derived cells, such as Marc-145. The latter is currently used for vaccine production. However, since virus entry in Marc-145 cells is different compared to entry in primary macrophages, specific epitopes associated with virus entry could potentially alter upon growth on Marc-145 cells. To avoid this, we constructed CHO and PK15 cell lines recombinantly expressing the PRRSV receptors involved in virus entry into macrophages, sialoadhesin (Sn) and CD163 (CHOSn-CD163 and PK15Sn-CD163) and evaluated their potential for production of PRRSV.

Results: Detailed analysis of PRRSV infection revealed that LV and VR-2332 virus particles could attach to and internalize into the CHOSn-CD163 and PK15Sn-CD163 cells. Initially, this occurred less efficiently for macrophage grown virus than for Marc-145 grown virus. Upon internalization, disassembly of the virus particles was observed. The two cell lines could be infected with PRRSV strains LV and VR-2332. However, it was observed that Marc-145 grown virus infected the cells more efficiently than macrophage grown virus. If the cells were treated with neuraminidase to remove cis-acting sialic acids that hinder the interaction of the virus with Sn, the amount of infected cells with macrophage grown virus increased. Comparison of both cell lines showed that the PK15Sn-CD163 cell line gave in general better results than the CHOSn-CD163 cell line. Only 2 out of 5 PRRSV strains replicated well in CHOSn-CD163 cells. Furthermore, the virus titer of all 5 PRRSV strains produced after passaging in PK15Sn-CD163 cells was similar to the virus titer of those strains produced in Marc-145 cells. Analysis of the sequence of the structural proteins of original virus and virus grown for 5 passages on PK15Sn-CD163 cells showed either no amino acid (aa) changes (VR-2332 and 07V063), one aa (LV), two aa (08V194) or three aa (08V204) changes. None of these changes are situated in known neutralizing epitopes.

Conclusions: A PRRSV susceptible cell line was constructed that can grow virus to similar levels compared to currently available cell lines. Mutations induced by growth on this cell lines were either absent or minimal and located outside known neutralizing epitopes. Together, the results show that this cell line can be used to produce vaccine virus and for PRRSV virus isolation.

Figure 1

Construction of CHO^Sn-CD163 and PK15^Sn-CD163 cell lines. A) Schematic representation of the construction of CHO^Sn-CD163 and PK15^Sn-CD163 cell lines To construct a cell line co-expressing Sn and CD163, CHO-K1 or PK15 cells were transfected with a plasmid containing the Sn cDNA and a geneticine resistance gene. The cells were single cell cloned and clones were screened for Sn expressing cells. After selection for geneticine resistance, the obtained CHO^Sn or PK15^Sn cells were transfected with a plasmid containing the CD163 cDNA and a zeocin resistance gene, which allowed selection of cells expressing both Sn and CD163. B) Immunofluorescence staining of the obtained CHO^Sn-CD163 or PK15^Sn-CD163 cells for Sn and CD163. Some CHO^Sn-CD163 clones (IF3, IC5 and ID9) and PK15^Sn-CD163 clones (IXA3 and IXH7) are represented with their Sn and CD163 expression.

Figure 2

Effect of cell density on susceptibility of CHO^Sn-CD163 and PK15^Sn-CD163 cells to PRRSV infection. CHO^Sn-CD163 and PK15^Sn-CD163 cells were cultivated for 2 days before they were inoculated with Marc-145 grown LV, Marc-145 grown VR-2332 or macrophage grown LV. The black bars represent a cell density of 100 000 cells/ml, the grey bars 200 000 cells/mL and the white bars 300 000 cells/ml. The graphs show the percentage of infected cells. Values represent mean ± SD of three experiments.

Figure 3

Attachment, internalization, disassembly and infection in CHO^Sn-CD163 and PK15^Sn-CD163 cells. CHO^Sn-CD163 clone IC5 and PK15^Sn-CD163 clone IXH7 were inoculated with Marc-145 grown LV, Marc-145 grown VR-2332 or macrophage grown LV. The different stages of the viral replication cycle were investigated by immunofluorescence staining of the virus.

Figure 4

PRRSV infection kinetics in CHO^Sn-CD163 and PK15^Sn-CD163 cells. CHO^Sn-CD163 and PK15^Sn-CD163 cells were inoculated with Marc-145 grown LV (open square), Marc-145 grown VR-2332 (black square) or macrophage grown LV (black circle). After 1, 2, 3, 5 and 7 dpi the cells were fixed and an immunoperoxidase staining was performed. The amount of infected cells were counted and expressed in the graphs as the percentage of infected cells. Values represent mean ± SD of three experiments.

Figure 5

Virus production on CHO^Sn-CD163 and PK15^Sn-CD163 cells. CHO^Sn-CD163 clone IC5 and PK15^Sn-CD163 clone IXH7 were inoculated with Marc-145 grown VR-2332 (black square), macrophage grown LV (open circle), macrophage grown 07V063 (black triangle), macrophage grown 08V204 (open triangle) or macrophage grown 08V194 (black diamond). The virus was passages 5 times and the supernatant was titrated. Values represent mean ± SD of three titrations.