Comparative infection efficiency of Porcine reproductive and respiratory syndrome virus field isolates on MA104 cells and porcine alveolar macrophages

Abstract

Isolation of Porcine reproductive and respiratory syndrome virus (PRRSV) on MA104 or MARC-145 cells is frequently used in PRRS diagnosis. However, the ability of recent field isolates to grow on these established simian cell lines has not been determined. The aim of this study was to characterize the growth of PRRSV field isolates on primary porcine alveolar macrophages (PAMs) and MA104 cells in comparison with the growth of the laboratory-adapted strain VR-2332. A cytopathic effect was observed in 70% of serum samples after 1 passage on PAMs and was verified by immunofluorescent staining or reverse transcriptase-polymerase chain reaction. Field isolate growth was observed on MA104 cells for only 1 of 50 serum samples after 14 d. Strain VR-2332 grew readily in MA104 cells [maximum titer, 10⁷ TCID₅₀ (median tissue culture infective dose) per milliliter at 30 h] but not in PAMs (10² TCID₅₀/mL at 72 h). These results show that PAMs are superior to simian cells for diagnostic isolation of current field PRRSV strains.

Porcine reproductive and respiratory syndrome virus (PRRSV), a member of the family Arteriviridae in the order Nidovirales, is a small, enveloped virus with a single-stranded positive-sense RNA genome. It causes a disease characterized by reproductive failure, including early stillbirth and late-term abortion, as well as respiratory distress in piglets and influenza-like disease in growing and finishing swine (1–4).

Since recognition of this virus, most PRRSV studies in North America have been carried out in continuous cell lines derived from green monkey kidney epithelial cells (MA104, CL2621, and MARC-145) (5–10). Although porcine alveolar macrophages (PAMs) are the naturally permissive host cell, they are used less, owing to the difficulty of isolating sufficient quantities for routine viral studies. During a study of genetic variation in field viruses, we observed that field strains grew better on PAMs than on MA104 cells. Therefore, we conducted a systematic study to characterize the growth of field isolates on primary PAMs and MA104 cells and developed a protocol for the isolation and storage of a large population of PAMs from 5- to 6-wk-old piglets for the isolation of field isolates.

Alveolar macrophages were obtained from seven 5- to 6-wk-old, cross-bred Yorkshire-Landrace male piglets that were free of Mycoplasma hyopneumoniae and PRRSV. The piglets were anesthetized with tiletamine hydrochloride–zolazepam hydrochloride (Telazol; Fort Dodge Laboratories, Fort Dodge, Iowa, USA), 8 mg/kg injected intramuscularly (IM), in combination with xylazine hydrochloride, 8 mg/kg injected IM, and subsequently were euthanized with Beuthanasia-D Special (Schering–Plough Animal Health, Union, New Jersey, USA), 0.5 mL/kg injected intravenously, in accordance with approved University of Minnesota Institutional Animal Care and Use Committee protocols.

The thoracic cavity was opened to remove the pulmonary and cardiac organs with the trachea clamped. The lungs were filled with 500 mL of sterile phosphate-buffered saline (PBS) containing gentamicin (Invitrogen, Carlsbad California, USA), 10 mg/mL. The lungs were gently massaged, and the lavage fluid was aspirated with a 25-mL pipette and collected in a sterile bottle. The collected fluid was always white, with foamy surfactant. The lavage was repeated twice and yielded a total volume of about 1 L.

The collected cell suspension was centrifuged at 500 × g for 10 min at 4°C. After 2 washes with PBS–gentamicin, the PAM pellet was suspended in 100 mL of RPMI 1640 with L-glutamine containing 5% fetal bovine serum (FBS), 4 mM HEPES, 1× minimum essential medium (MEM) nonessential amino acids, and 1 mM sodium pyruvate. To estimate the total amount of viable PAMs collected, 10 μL of the final cell suspension was diluted in 90 μL of 10% trypan blue in PBS, and the cells were counted with a hemocytometer (11,12).

The PAMs were stored frozen in cryogenic vials (Nalgene Cryoware; Fisher Scientific, Pittsburgh, Pennsylvania, USA) in a cryoprotective medium containing 20% FBS and 5% dimethylsulfoxide in complete RPMI 1640. The concentration was adjusted to 2 × 10⁷ cells in 1.5 mL of the medium. The cells were cooled slowly through several steps inside a box of polystyrene foam 1 to 2 cm thick, starting at 4°C for 1 to 2 h, then −20°C for 1 to 2 h, and finally −80°C overnight; they were then transferred to liquid nitrogen (13).

The PAMs and MA104 cells [clone of embryonic kidney epithelial cells from the African green monkey (Cercopithecus aethiops); American Type Culture Collection catalog no. CRL-378.1] were used to isolate PRRSV from field samples. The MA104 cells were cultured in MEM with 2 mM L-glutamine and Earle’s basic salt solution adjusted to contain 1.5 g/L of sodium bicarbonate, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, and 10% FBS.

The PAMs were thawed in a 37°C water bath, washed twice with sterile PBS, suspended in RPMI medium with 5% FBS at a concentration of 2 × 10⁶ cells per well in a 24-well plate, and then incubated at 37°C in CO₂ in a humidified chamber. After 24 h, nonadherent cells were removed, fresh medium was added, and the cultures were tested for bacterial contamination (6).

Serum samples from 50 clinically ill pigs in PRRS outbreaks at finisher and sow farms in the north-central region of the United States were used. Virus growth was quantified as the median tissue culture infective dose per milliliter (TCID₅₀/mL) (14) by means of quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Viral RNA was extracted from cell culture supernatants and infected cells with the RNeasy Mini Kit (Qiagen, Valencia, California, USA) according to the manufacturer’s instructions. Quantitative RT-PCR was performed with the TaqMan One-Step RT-PCR kit (Applied Biosystems, Foster City, California, USA) to amplify the open reading frame 7-3′-untranslated region (15).

The RT-PCR reactions contained 2 μL of the RNA sample, 10 μL of 2 × master mix, forward primer (300 nM final), reverse primer (300 nM final), TaqMan probe (400 nM final), 0.5 μL of MultiScribe/RNase inhibitor (Applied Biosystems), and 5.3 μL of DNase/RNase-free water, in a total volume of 20 μL. Triplicate reactions were processed in an ABI Prism 7500 (Applied Biosystems) for 30 min at 50°C for reverse transcription, followed by 10 min at 95°C and 45 cycles of 15 s at 95°C and 1 min at 60°C.

The PAMs and MA104 cells were seeded onto 24-well plates at concentrations of 2 × 10⁶ and 2 × 10⁵ per well, respectively. The cells were inoculated with 300 μL of the test serum sample. The laboratory-adapted strain VR-2332 was used as a positive control at a multiplicity of infection (MOI) of 2. After 1 h of virus adsorption at room temperature on a rocker, fresh medium was added, and the cells were cultured at 37°C in a CO₂ incubator. Supernatant and cells were collected when a cytopathic effect (CPE) was observed in 70% or more of the culture well or after 6 d if no CPE was observed. Virus isolation was confirmed by RT-PCR and immunofluorescence (IF) (16).

For IF the cells were scraped from the well, air-dried on glass slides, fixed with cold methanol, washed gently with PBS (pH 7.2), and incubated overnight at 4°C with fluorescein-conjugated monoclonal antibody SDOW-17F (Rural Technologies, Brookings, South Dakota, USA), which is specific for the viral nucleocapsid. The reaction was observed on an Olympus 1X70 inverted fluorescence microscope (Olympus America, Center Valley, Pennsylvania, USA) (17).

The lung lavage in the 7 piglets generated an average of 2.3 × 10⁹ PAMs in 100 mL (Table I), 90% of which were viable by trypan blue exclusion and 70% of which were adherent alveolar macrophages. The remaining cells were nonadherent macrophages, lymphocytes, and epithelial cells.

Table I.

Total cell yields in pulmonary lavage fluid from 7 piglets

Pig ID	PBS wash (mL)	Fluid recovered (mL)	Cells/mLa
1	1000	600	2 × 107
2	1000	750	8 × 107
3	1000	700	2 × 107
4	1000	500	1 × 107
5	1000	650	2 × 107
6	1000	750	3 × 107
7	1000	700	5 × 106
Average	1000	665	2.3 × 107

PBS — phosphate-buffered saline.

^a After washing, cells were resuspended in 100 mL of PBS. The total average number of cells collected was 2.3 × 10⁷ × 100 mL, or 2.3 × 10⁹. All batches of lavage cells collected supported the growth of Porcine reproductive and respiratory syndrome virus (PRRSV).

When PAMs were used to isolate PRRSV, 20 field strains were isolated as early as 72 h after inoculation, and 15 additional strains were isolated when the PAMs were incubated for up to 6 d. Only 1 field strain was isolated when MA104 cells were inoculated and incubated for 14 d. Virus aggregates were observed in PAMs by IF (Figure 1). Morphologic changes in cells started to appear 48 h after inoculation. After 6 d, wells showed a CPE of more than 70%. Of the 50 serum samples, PRRSV strains were isolated from 35 (70%) after 6 d of culture; 18 samples (36%) were IF-positive at 2 d, and 33 (66%) were RT-PCR-positive at 6 d (Table II).

Figure 1.

Immunofluorescent staining of PRRSV infection in MA104 cells (A) and porcine alveolar macrophages (PAMs) (B) inoculated with field isolates of Porcine reproductive and respiratory syndrome virus (PRRSV). Viral nucleocapsid was visualized with monoclonal antibody SDOW-17F conjugated with fluorescein (green). The red represents counterstaining with Evan’s blue.

Table II.

Results of attempts to isolate field strains of PRRSV from serum collected from farms with chronic PRRS on MA104 cells and porcine alveolar macrophages (PAMs)

	Cell type; time after inoculation of test serum; result
	MA104; 14 d		PAM; 6 d		PAM; 48 h		PAM; 6 d
Serum samples	CPE	No CPE	CPE	No CPE	IF +	IF −	Ct < 38 (+)	Ct > 38 (−)
No.	1	49	35	15	18	32	33	17
%	2	98	70	30	36	64	66	34

CPE — cytopathic effect; IF — immunofluorescence with fluorescein-conjugated monoclonal antibody SDOW-17F; Ct — cycle threshold value, representing the amount of viral nucleic acid in the culture medium, as measured by quantitative reverse transcriptase-polymerase chain reaction.

The amount of VR-2332 obtained from inoculated MA104 cells was 10⁷ TCID₅₀/mL [cycle threshold (Ct) = 18 to 20] after 30 h and remained constant for several days (Figure 2B, open circles). By contrast, VR-2332 growth on PAMs was negligible (Ct = 35 to 36) (Figure 2A, open circles). When MA104 cells were inoculated with serum samples from field cases of PRRS, growth was not observed (Ct = 36 to 39), whereas the amount of virus after 72 h of incubation on PAMs was 10^6.5 TCID₅₀/mL (Ct = 19) (Figures 2A and 2B, closed circles).

Figure 2.

Growth of the laboratory-adapted strain VR-2332 and a field isolate on alveolar macrophages (A) and MA104 cells (B). Growth was determined by the amount of viral nucleic acid in the culture medium, as measured by quantitative reverse transcriptase-polymerase chain reaction, and is shown as the cycle threshold (Ct) value. For VR-2332 (open circles) the maximum titer was at 30 h in MA104 cells (panel B). The field isolate (closed circles) reached the same Ct value at 72 h on PAMs (panel A).

Traditionally, isolation of PRRSV field strains from serum samples in North America has been performed in MA104 cells. However, most of these strains were unable to grow, even though the samples came from endemically infected sow herds. Similar results were observed early in the history of PRRS. Bautista et al (6) reported differences in the isolation of PRRSV on MA104 cells (also known as CL2621 cells) and PAMs from serum samples collected from animals suspected of having PRRS. They observed field isolates that were cultured only on MA104 cells, only on PAMs, or on both cell types. They concluded that although PAMs were relatively more sensitive for PRRSV isolation, both PAMs and CL2621 cells should be used. We observed substantial differences in PRRSV infection efficiency when comparing the growth of recent PRRSV field isolates on MA104 cells and PAMs. At 6 d after inoculation, 70% of the field PRRSV strains had grown in PAMs versus 2% in MA104 cells. By 2 d after inoculation, 36% of the isolates showed evidence of protein expression by IF, and within 6 d 66% had produced progeny virus according to the results of RT-PCR analysis of media supernatants. We also observed that the widely used laboratory reference strain VR-2332 grew efficiently in MA104 cells, but its growth was severely retarded in PAMs.

The isolation results indicate that the virus present in the serum samples was able to grow productively on PAMs. Breed variation in the source of PAMs is reported to influence the ability of PRRSV to grow in vitro on PAMs: alveolar macrophages from Landrace sows poorly support the growth of PRRSV (18). The genetic composition of the 7 piglets from which the PAMs were obtained for our study is not known. Interestingly, we found that wild-type viruses appeared not to recognize MA104 cells as permissive. Growth on these cells seems to require conditions of incubation or viral features that are not always present. This finding also indicates that the presence of CD163, the PRRSV receptor, is not by itself sufficient for efficient infection of host cells (19).

For a better rate of isolation of wild-type PRRSV from field samples, it is important to use appropriate aseptic techniques for collection. We were unable to evaluate samples that were highly contaminated and that required special treatments to achieve sterility, which may have reduced the sensitivity of virus isolation.

A large population of alveolar macrophages was obtained from the lungs of young pigs, and all samples supported PRRSV growth after freezing and thawing. More than two-thirds of the cells were adherent, and the viability was approximately 90% after thawing.

In conclusion, the results of this study indicate that frozen alveolar macrophages, but not MA104 cells, or the subclone MARC-145, are suitable host cells for routine isolation of PRRSV field strains.