Susceptibility of porcine pulmonary microvascular endothelial cells to porcine reproductive and respiratory syndrome virus

Abstract

Microvascular endothelial cells possess versatile functions and their roles in a variety of viral infections have been documented. Porcine reproductive and respiratory syndrome virus (PRRSV) infection induces severe lung inflammatory lesions in piglets, which is manifested as pulmonary endothelial dysfunction. However, the underlying mechanism of PRRSV affecting porcine pulmonary microvascular endothelial cells (PMECs) remains unknown. This study aimed to evaluate the susceptibility of PMECs to PRRSV. Primary PMECs were isolated and purified from piglet lungs, and the expression of three PRRSV receptors was characterized using immunofluorescence. Overt cytopathic effects of the PRRSV strain HN in PMECs were observed at day five post-infection, and PRRSV antigens in PMECs were determined at both RNA and protein levels using immunofluorescence and quantitative RT-PCR assays. The viral antigen significantly increased at 96 hr post-infection, and infectious virus was recovered from the supernatant of the infected PMECs. The results show that PMECs can be infected with the PRRSV strain HN, and that their receptor expression pattern is different from that of alveolar macrophages. The results of this study shed light on the potential roles of PMECs in PRRSV infection and provide a comprehensive understanding of the pathogenesis underlying its severe manifestation.

Keywords: cytopathic effects; infection; microvascular endothelial cells; porcine reproductive and respiratory syndrome virus; receptor.

Fig. 1.

Morphological observation and identification using PECAM-1 immunofluorescence of porcine pulmonary microvascular endothelial cells (PMECs). (A) PMECs from piglet lungs were observed under a light microscope; (B) Porcine PMECs of passage 3 were stained with the anti-PECAM-1 antibody at 24 hr after cultivation; (C) Negative control of PECAM-1 immunofluorescence; Bar=100 µm.

Fig. 2.

Identification of porcine reproductive and respiratory syndrome virus receptors in porcine pulmonary microvascular endothelial cells using immunofluorescence staining. Cells were seeded into 24-well plates and fixed after 24 hr of incubation. Indirect immunofluorescence was performed using anti-CD151 (A), anti-CD163 (B), and anti-CD169 antibodies (C). Bar=100 µm.

Fig. 3.

Observation of cytopathic effects (CPE) in porcine reproductive and respiratory syndrome virus strain HN-infected porcine pulmonary microvascular endothelial cells. Cells were sub-cultured on 24-well plates and infected or mock-infected with PRRSV strain HN. Cellular morphology was observed and photographs were taken every day. Bar=100 µm. DPI: days post-infection.

Fig. 4.

Immunofluorescence detection of viral antigens in porcine reproductive and respiratory syndrome virus (PRRSV)-infected porcine pulmonary microvascular endothelial cells. Cells were seeded into 24-well plates for 12 hr and infected with the PRRSV strain HN. Staining for PRRSV GP5 protein and N protein was carried out at 48 hpi. Bar=100 µm. hpi: hours post-infection. MARC-145 cells: African green monkey embryonic renal epithelial cells.

Fig. 5.

Determination of porcine reproductive and respiratory syndrome virus (PRRSV) replication kinetics in porcine pulmonary microvascular endothelial cells by using qRT-PCR assay. (A) Agarose gel electrophoresis analysis of the PCR products. Lane M: DL2000 marker, lane 1: β-Actin, lane 2: negative control of β-Actin, lane 3: PRRSV N protein, lane 4: negative control of PRRSV N protein. (B) Relative ratios of PRRSV N protein gene expression to β-Actin. * denotes a significant difference (P<0.05) between 48 and 24 hpi; ** denotes an extremely significant difference (P<0.01) between 96 and 24 hpi.

Fig. 6.

Replication efficiencies of porcine reproductive and respiratory syndrome virus (PRRSV) in porcine pulmonary microvascular endothelial cells (PMECs) MARC-145 cells at different time points post-infection. Porcine PMECs were seeded on 6-well plates and infected with PRRSV strain HN. At 12, 24, 48, 72 and 96 hpi, cell supernatant was collected and titrated on MARC-145 cells. ** and ## denote extremely significant differences (P<0.01) compared to PMECs and MARC-145 cells at 12 hpi, respectively, and §§ denotes extremely significant differences (P<0.01) between PMECs and MARC-145 cells at the same time points.