Abstract
BackgroundPorcine 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.ResultsDetailed 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.ConclusionsA 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.
Highlights
Porcine reproductive and respiratory syndrome virus (PRRSV) causes major economic losses in the pig industry worldwide
The results show that this cell line can be used to produce vaccine virus and for PRRSV virus isolation
Effect of cell density and cultivation time of CHOSn-CD163 and PK15Sn-CD163 cells on the susceptibility to PRRSV infection To determine the effect of cell density and cultivation time of the cells on susceptibility to PRRSV infection, 3 CHOSn-CD163 cell clones (IC5, ID9 and IF3) and 2 PK15SnCD163 cell clones (IXH7 and IXA3) were seeded at different cell densities (100 000, 200 000 or 300 000 cells/mL) and inoculated with 50 μL containing 104 TCID50 Marc145 grown Lelystad virus (LV), Marc-145 grown VR-2332 or macrophage grown LV at different days post seeding (1, 2 or 3 days post seeding)
Summary
Porcine reproductive and respiratory syndrome virus (PRRSV) causes major economic losses in the pig industry worldwide. 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. Porcine reproductive and respiratory syndrome virus (PRRSV) is a member of the family Arteriviridae, order Nidovirales [1,2] causing major economic losses in the pig industry worldwide [3]. Efficient PRRSV replication is only observed in primary pig macrophages (e.g. alveolar macrophages) [14], differentiated monocytes [15] or African green monkey kidney derived cells, such as Marc-145 [14,16]. CD163 is essential for PRRSV infection of Marc-145 cells, but its role in this process is still unclear [24]
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