PRRSV Propagation, Isolation, and Virus Titer Determination (Obj. 4)

The PRRSV used in this experiment (MN-184, PRRSV-2 Lineage 1) was provided by the Veterinary Diagnostic Laboratory (College of Veterinary Medicine, Iowa State University). The virus was propagated in the MARC-145 cell line, a clone of the African monkey kidney cell line MA-104 (Kim et al., 1993). MARC-145 cells were cultured in the RPMI-1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 0.05 mg/ml gentamicin, 100 unit/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin. Large-scale virus propagation was conducted in five-layer flasks (Thermo Fisher Scientific, Rochester, NY, United States). Briefly, when cells reached 80–90% confluence, stock PRRSV was added. The estimated number of cells at that range of confluency was 18.6–21.0 × 10⁶ cells/layer (Thermo Fisher Scientific – US, 2021). Flasks were observed daily for cytopathic effect (CPE). When the CPE was abundant (5–7 days), flasks underwent two freeze-thaw cycles, followed by centrifugation at 4,200×g for 10 min to harvest the supernatant. Approximately 3 L of virus inoculum was harvested with a geometric mean virus titer of 10^5.56 TCID₅₀/ml. The inoculum was thoroughly mixed to ensure homogeneity, distributed into 30 ml aliquots and stored at −80°C.

For determination of TCID₅₀ in research samples, impinger fluid was transferred to a biosafety cabinet in a BSL-2 laboratory, and tenfold serial dilutions were performed, with eight replicates for each sample. Each well (in 96-well plates) was prefilled with 270 μl of RPMI-1640 medium, and then the sample was added to the first row of the plates; the solution was mixed, and then 30 μl of liquid transferred sequentially from one row to another. Thus, the dilution for each row ranged from 10⁰, 10^–1, …, to 10^–7, respectively. We considered the 10⁰ dilutions to improve method detection limits for the benefit of the subsequent experiments on PPRSV survival after UV treatment. Thereafter, 100 μl from each well was inoculated into 80∼90% subconfluent MARC-145 cells grown in 96-well plates. The plates were incubated at 37°C in a humidified 5% CO₂ incubator. CPE development was checked under a microscope daily, and infected wells were marked as positive until no more additional wells were identified as infected (5–7 days). To confirm the presence of PRRSV, the plates were fixed (80% acetone for 10 min), dried, and stained with a PRRSV nucleocapsid protein-specific monoclonal antibody (SDOW17-F) conjugated to fluorescent isothiocyanate (Rural Technologies, Inc., Brookings, SD) for 1 h at 37°C. The antibody conjugate was decanted, and the cell plates were washed with PBS (1× pH 7.4) three times, 5 min each time. Plates were read under an Olympus IX71 fluorescent microscope (Olympus America Inc., Center Valley, PA, United States). The Spearman-Karber method (Karber, 1931; Lei et al., 2020) was used to calculate the virus titers, which were based on the number of wells showing positive PRRSV-specific fluorescence at specific dilution and the results expressed as TCID₅₀ per milliliter of the impinger fluid (Cutler et al., 2012; Yim-Im et al., 2021).