Comparison of ZMAC and MARC-145 Cell Lines for Improving Porcine Reproductive and Respiratory Syndrome Virus Isolation from Clinical Samples

Abstract

The MARC-145 cell line is commonly used to isolate porcine reproductive and respiratory syndrome virus (PRRSV) for diagnostics, research, and vaccine production, but it yields frustratingly low success rates of virus isolation (VI). The ZMAC cell line, derived from porcine alveolar macrophages, has become available, but its utilization for PRRSV VI from clinical samples has not been evaluated. This study compared PRRSV VI results in ZMAC and MARC-145 cells from 375 clinical samples (including 104 lung, 140 serum, 90 oral fluid, and 41 processing fluid samples). The PRRSV VI success rate was very low in oral fluids and processing fluids regardless of whether ZMAC cells or MARC-145 cells were used. Success rates of PRRSV VI from lung and serum samples were significantly higher in ZMAC than in MARC-145 cells. Lung and serum samples with threshold cycle (C_T ) values of <30 had better VI success. PRRSV-2 in genetic lineages 1 and 8 was isolated more successfully in ZMAC cells than in MARC-145 cells, whereas PRRSV-2 in genetic lineage 5 was isolated in the two cell lines with similar success rates. For samples with positive VI in both ZMAC and MARC-145 cells, 14 of 23 PRRSV-2 isolates had similar titers in the two cell lines. A total of 51 of 95 (53.7%) ZMAC-obtained PRRSV-2 or PRRSV-1 isolates grew in MARC-145 cells, and all 46 (100%) MARC-145-obtained isolates grew in ZMAC cells. In summary, ZMAC cells allow better isolation of a wide range of PRRSV field strains; however, not all of the ZMAC-obtained PRRSV isolates grow in MARC-145 cells. This report provides important guidelines to improve isolation of PRRSV from clinical samples for further characterization and/or for producing autogenous vaccines.

Keywords: MARC-145; ZMAC; genetic lineage; porcine reproductive and respiratory syndrome virus; virus isolation.

FIG 1

Immunofluorescence staining of PRRSV in ZMAC and MARC-145 cells. Examples of direct immunofluorescence staining of mock- and PRRSV-infected MARC-145 and ZMAC cells, using FITC-conjugated PRRSV monoclonal antibodies, are depicted in panels A, B, E, and F. Examples of indirect immunofluorescence staining of mock- and PRRSV-infected MARC-145 and ZMAC cells, using unconjugated PRRSV monoclonal antibodies and FITC-conjugated goat anti-mouse secondary antibody, are depicted in panels C, D, G, and H.

FIG 2

Comparison of infectious titers (TCID₅₀/ml) of 23 PRRSV-2 isolates obtained in ZMAC and MARC-145 cells. The same sets of 23 clinical samples were VI positive in both ZMAC and MARC-145 cells. The isolates obtained in ZMAC cells were titrated in ZMAC cells, and the isolates obtained in MARC-145 cells were titrated in MARC-145 cells. Six isolates that had higher titers in ZMAC cells than in MARC-145 cells are boxed with a dashed line. Fourteen ZMAC and MARC-145 isolates that had similar titers are boxed with a solid line. The lineage (e.g., L1) and RFLP pattern (e.g., 1-7-4) of each isolate are also indicated.