Physiological and immunological responses of porcine primary endometrial cells to porcine reproductive and respiratory syndrome (PRRS) virus infection

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) is highly limited to only cell subsets that express PRRSV receptors. Reproductive organ revealing the typical signs of PRRSV infection may be the critical site of problem syndromes. Persistent PRRSV producing re-infection via horizontal or vertical transmission could not be eradicated from herds. This research examined the possibility of porcine endometrium to be a PRRSV permissive cell and serves as the primary cause of the persistent PRRSV. Cellular and immunological in response to PRRSV relevant to viral replication, shedding and re-circulation was assessed. The different outcomes between the different genotypes (type I vs. II), and routes of infection (apical vs. basolateral) were compared. Porcine glandular endometrial epithelial cells (PE) isolated from 4-6 months old PRRSV-free pre-puberty gilts (n=5 pigs) were cultured in standard medium DMEM with 5% fetal bovine serum until 90% confluent. Fresh isolated PRRSV type I or type II (at TCID100/2 ml) were inoculated to apical or basolateral membrane of PE for 1 hr. The occurrence of cytopathic effect (CPE) were observed daily. PRRSV-positive cells and cellular PRRSV mediator proteins, CD151, CD163, sialoadhesin (Sn), integrin and vimentin, were quantified by immunohistochemistry (IHC). Related cytokine secretion, CCL2, IL-1β, IL-6, IL-8, IFN-g and TNF-α was measured by enzyme linked immunosorbent assay (ELISA), at 0, 2, 4 and 6 day-post-infection (dpi). The mRNA expression of PRRSV mediator, toll-like receptors (TLRs) and cytokines were evaluated by real-time RT-PCR. Effects of PRRSV re-infection in modulating all the responses of primary infection were considered by repeating the infection at 4 dpi at the same PE. At early stage of infection, at 4dpi, CPE along and PRRSV proteins was observed in apical PRRSV infected PE, but was observed later in basolateral-infected PE. Infection with type II produced these infectivity effects rather than type I (p<0.05). Prior to infection, mediator proteins CD151, Sn, integrin and vimentin but not CD163 were expressed. Type I up-regulated CD151, CD163, Sn and integrin mRNA higher than mock and type II (p<0.05). Changes of mediator proteins were observed differently during 4-6 dpi (p<0.05). Apical infection with type I up-regulated all mediators except Sn, whereas type II down-regulated Sn, integrin and vimentin. Basolateral type I and II infection down-regulated integrin and vimentin (p<0.05), but up-regulated CD151, CD163 and Sn (p<0.05). Up-regulation of TLR1/TLR3 and TLR10 were induced by primary infection with type I and II, respectively. All primary infection down-regulated TLR4 mRNA (p<0.05). Re-infection with PRRSV particularly type I up-regulated the level of TLR1, TLR2 and TLR4 expression (p<0.05). Down-regulation of TLR5 and TLR8 were later observed in primary or re-infected PE cells (p<0.05). Re-infection with type I or II completely decreased IL-6 mRNA, but not other genes. Primary PRRSV infection could not alter CCL2, IL1β, IL-8 and IFN-g secreted by PE , but type I infection increased IL-6 secretion (p<0.05). Primary or re-infection with PRRSV type I or II dampened TNF-α secretion significantly (p<0.05). Noticeably, in the present study, supernatant collected from all PRRSV-infected cells contains PRRSV at TCID100/ml throughout the study. In summary, endometrial cells are susceptible to either apical and basolateral PRRSV infection, and long-lasting re-circulate PRRSV. Effects of primary infection may be mediated by TLRs or mediators. Modification in the synthesis of TLRs, PRRSV mediators and cytokines by PRRSV could be enhanced or suppressed depending on time course, genotype or route of infection. PRRSV type II has more virulence than type I but PRRSV type I produced more to susceptibility for executive infection. Therefore, direct PRRSV infection to PE cells may play a role in PRRSV-induced reproductive failure and may be the cause of persistent PRRSV infection in sow.