Effects and mechanisms of porcine reproductive and respiratory syndrome virus (PRRSV) type 1 and type 2 on the permeability and viability of porcine endometrial epithelial cells

Abstract

Both PRRSV types 1 and 2 revealed the sign of reproductive disorders associated with a lesion at implantation sites. Impairment of maternal glandular endometrium cell integrity and function by PRRSV infection may impact a proper nourishing fetus. This research was aimed to examine the effects of PRRSV type 1 and type 2 directly on the viability and barrier function of the endometrium using porcine glandular endometrial epithelial cell culture (PEG). The comparison of the route and the strain of PRRSV infection coinciding with their virulent in reproductive epithelia were considered. PEG cells isolated from the 4-6 months old PRRSV-free-herd gilts (n=7 pigs) were cultured in standard media DMEM with 5% fetal bovine serum until 90% confluent. The fresh isolated PRRSV type 1 and type 2 (at TCID100/2 ml) inoculated to either apical or basolateral site of PEG cells for 1 h. The cytopathic effect (CPE) was observed daily. The permeability assessment of barrier function, the measurement of FITC dextran 4 kDa (FD-4) flux and transepithelial electrical resistance (TER), were performed at 0, 2, 4 and 6 days post infection (dpi). The expression of TJ protein genes; Cldn 1, 2, 3, 4, 5, 7, 8 and ZO-1 were detected by real-time qPCR and normalized with GAPDH at 4 dpi. The viability was determined by MTT assay and annexin V/propidium iodide (PI) assay at 0, 2, 4 and 6 dpi. All PRRSV inoculations to PEG cells produced a various CPE formation upon the inoculation. Apical inoculation with PRRSV type 2 decreased TER and increased FD-4 flux at 4 dpi (p<0.05). This indicates the cytotoxicity of PRRSV type 2 infection. In contrast, basolateral inoculation of PRRSV type 1 and 2 had no effects but maintained the integrity of the TJ barrier reflected by the stable of FD-4 flux through 6 dpi (p<0.05). Both viruses seem to increase the barrier function by up-regulating the barrier builder TJ Cldn5 but down-regulating the pore-forming TJ Cldn7 (p<0.05). The additional TJs gene was differently a target of PRRSV type 1 and 2 infections; Cldn3 and Cldn8 for PRRSV type 1 vs. ZO-1 for PRRSV type 2. However, the alteration of TJs expression induced by PRRSV was not relevant to their effects on TJs barrier functions. In the viability test, PRSSV type 2 increased cell proliferation, although the necrotic cell was concomitantly detected at 2 and 4 dpi (p<0.05). A few necrotic cells were also founded by PRRSV type 1 infection at 2 dpi. All of the cytotoxicity or proliferative effects were recovered at 6 dpi in all PRRSV infected PEG. However, whether the recovery PEG cell has a proper function is suspicious. The conclusion from our findings is PRRSV type 2 has more severity on the TJs barrier leaky and cell death than type 1 PRRSV in porcine endometrium. Since PRRSV can disturb TJs mRNA expression associated with necrosis, mucosal contamination or inoculation with PRRSV may be at risk or associated with PRRSV-induced reproductive disorders.