Comparison of Primary Virus Isolation in Pulmonary Alveolar Macrophages and Four Different Continuous Cell Lines for
Jiexiong Xie1, Nick Vereecke1,2, Sebastiaan Theuns2
1Laboratory of Virology, Faculty of Veterinary Medicine, Ghent University, 9820 Merelbeke, Belgium.
Abstract:
Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) has a highly restricted cellular tropism. In vivo, the virus primarily infects tissue-specific macrophages in the nose, lungs, tonsils, and pharyngeal lymphoid tissues. In vitro however, the MARC-145 cell line is one of the few PRRSV susceptible cell lines that are routinely used for in vitro propagation. Previously, several PRRSV non-permissive cell lines were shown to become susceptible to PRRSV infection upon expression of recombinant entry receptors (e.g., PK15Sn-CD163, PK15S10-CD163). In the present study, we examined the suitability of different cell lines as a possible replacement of primary pulmonary alveolar macrophages (PAM) cells for isolation and growth of PRRSV. The susceptibility of four different cell lines (PK15Sn-CD163, PK15S10-CD163, MARC-145, and MARC-145Sn) for the primary isolation of PRRSV from PCR positive sera (both PRRSV1 and PRRSV2) was compared with that of PAM. To find possible correlations between the cell tropism and the viral genotype, 54 field samples were sequenced, and amino acid residues potentially associated with the cell tropism were identified. Regarding the virus titers obtained with the five different cell types, PAM gave the highest mean virus titers followed by PK15Sn-CD163, PK15S10-CD163, MARC-145Sn, and MARC-145. The titers in PK15Sn-CD163 and PK15S10-CD163 cells were significantly correlated with virus titers in PAM for both PRRSV1 (p < 0.001) and PRRSV2 (p < 0.001) compared with MARC-145Sn (PRRSV1: p = 0.22 and PRRSV2: p = 0.03) and MARC-145 (PRRSV1: p = 0.04 and PRRSV2: p = 0.12). Further, a possible correlation between cell tropism and viral genotype was assessed using PRRSV whole genome sequences in a Genome-Wide-Association Study (GWAS). The structural protein residues GP2:187L and N:28R within PRRSV2 sequences were associated with their growth in MARC-145. The GP5:78I residue for PRRSV2 and the Nsp11:155F residue for PRRSV1 was linked to a higher replication on PAM. In conclusion, PK15Sn-CD163 and PK15S10-CD163 cells are phenotypically closely related to the in vivo target macrophages and are more suitable for virus isolation and titration than MARC-145/MARC-145Sn cells. The residues of PRRSV proteins that are potentially related with cell tropism will be further investigated in the future.
Insights
New cell lines, PK15Sn-CD163 and PK15S10-CD163, show promise for Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) research. These cells better mimic in vivo conditions than MARC-145 cells, improving PRRSV isolation and growth.
Area of Science:
- Virology
- Cell Biology
- Animal Health
Background:
- Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) exhibits restricted cellular tropism, primarily infecting macrophages in vivo.
- MARC-145 cells are commonly used for in vitro PRRSV propagation, but their suitability as a replacement for primary pulmonary alveolar macrophages (PAM) is limited.
- Previous studies demonstrated that engineered cell lines expressing recombinant entry receptors can become susceptible to PRRSV infection.
Purpose of the Study:
- To evaluate the suitability of different cell lines, including engineered PK15 cells, as replacements for PAM in PRRSV isolation and growth.
- To compare the susceptibility of PK15Sn-CD163, PK15S10-CD163, MARC-145, and MARC-145Sn cells for primary PRRSV isolation from clinical samples.
- To identify potential correlations between viral genotype and cell tropism.
Main Methods:
- PRRSV isolation and titration were performed using five cell types: PAM, PK15Sn-CD163, PK15S10-CD163, MARC-145, and MARC-145Sn.
- Fifty-four field samples were sequenced to analyze viral genotypes.
- Genome-Wide Association Study (GWAS) was employed to correlate viral genomic sequences with observed cell tropism.
Main Results:
- PAM yielded the highest mean virus titers, followed by PK15Sn-CD163 and PK15S10-CD163 cells.
- Virus titers in PK15Sn-CD163 and PK15S10-CD163 cells showed significant correlation with PAM titers for both PRRSV1 and PRRSV2.
- Specific amino acid residues in PRRSV structural proteins (GP2, N, GP5) and non-structural proteins (Nsp11) were associated with differential growth in PAM and MARC-145 cells.
Conclusions:
- PK15Sn-CD163 and PK15S10-CD163 cells are phenotypically similar to in vivo target macrophages and are more suitable for PRRSV isolation and titration than MARC-145/MARC-145Sn cells.
- The study identified specific viral protein residues potentially influencing PRRSV cell tropism.
- Further investigation into these residues is warranted to understand PRRSV-host cell interactions.
