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

Delrue I, Van Gorp H, Van Doorsselaere J, Delputte PL, Nauwynck HJ - BMC Biotechnol. (2010)

Bottom Line: Comparison of both cell lines showed that the PK15Sn-CD163 cell line gave in general better results than the CHOSn-CD163 cell line.None of these changes are situated in known neutralizing epitopes.Mutations induced by growth on this cell lines were either absent or minimal and located outside known neutralizing epitopes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Virology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium.

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.

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Effect of cell density on susceptibility of CHOSn-CD163 and PK15Sn-CD163 cells to PRRSV infection. CHOSn-CD163 and PK15Sn-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.
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Figure 2: Effect of cell density on susceptibility of CHOSn-CD163 and PK15Sn-CD163 cells to PRRSV infection. CHOSn-CD163 and PK15Sn-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.

Mentions: For Marc-145 grown virus infection of CHOSn-CD163 cells, little difference was observed between different cell densities and days post seeding, although a density of 200 000 cells/mL and inoculation at 2 days post seeding seemed a little more efficient. The VR-2332 strain infected the CHOSn-CD163 cells more efficiently than the LV strain. For PK15Sn-CD163 cells, the infection rate for Marc-145 grown LV as well as VR-2332 was approximately 80%, independently of densities and cultivation time. CHOSn-CD163 clones IC5, ID9 and IF3 were equally sensitive to virus infection. There was no difference in sensitivity for infection between PK15Sn-CD163 clones IXH7 and IXA3. The PK15Sn-CD163 cells could be infected more efficiently than the CHOSn-CD163 cells (Figure 2).


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

Delrue I, Van Gorp H, Van Doorsselaere J, Delputte PL, Nauwynck HJ - BMC Biotechnol. (2010)

Effect of cell density on susceptibility of CHOSn-CD163 and PK15Sn-CD163 cells to PRRSV infection. CHOSn-CD163 and PK15Sn-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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2908558&req=5

Figure 2: Effect of cell density on susceptibility of CHOSn-CD163 and PK15Sn-CD163 cells to PRRSV infection. CHOSn-CD163 and PK15Sn-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.
Mentions: For Marc-145 grown virus infection of CHOSn-CD163 cells, little difference was observed between different cell densities and days post seeding, although a density of 200 000 cells/mL and inoculation at 2 days post seeding seemed a little more efficient. The VR-2332 strain infected the CHOSn-CD163 cells more efficiently than the LV strain. For PK15Sn-CD163 cells, the infection rate for Marc-145 grown LV as well as VR-2332 was approximately 80%, independently of densities and cultivation time. CHOSn-CD163 clones IC5, ID9 and IF3 were equally sensitive to virus infection. There was no difference in sensitivity for infection between PK15Sn-CD163 clones IXH7 and IXA3. The PK15Sn-CD163 cells could be infected more efficiently than the CHOSn-CD163 cells (Figure 2).

Bottom Line: Comparison of both cell lines showed that the PK15Sn-CD163 cell line gave in general better results than the CHOSn-CD163 cell line.None of these changes are situated in known neutralizing epitopes.Mutations induced by growth on this cell lines were either absent or minimal and located outside known neutralizing epitopes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Virology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium.

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.

Show MeSH
Related in: MedlinePlus