The porcine trophoblast cell line PTr2 is susceptible to porcine reproductive and respiratory syndrome virus-2 infection.

INTRODUCTION: Porcine reproductive and respiratory syndrome virus-2 (PRRSV-2) breaches the maternal-fetal interface (MFI) to infect porcine fetuses, yet the exact mechanism(s) of transmission is not understood. The objective of this study was to determine the susceptibility of porcine trophoblast cell line (PTr2) to PRRSV-2 infection to understand the potential role of the trophoblast in viral transmission to fetuses in vivo. METHODS: PTr2 cells were exposed in vitro to PRRSV-2 and then subjected to immunofluorescence analysis (IF), flow cytometry (FCM), real-time quantitative PCR (RT-qPCR), transmission electron microscopy (TEM) and immunogold electron microscopy (IEM) to assess viral infection. The effects of PRRSV-2 on PTr2 cell cycle progression and apoptosis, as well as the ability of PTr2 cells to produce infectious viral particles were also examined. RESULTS: PRRSV-2 was readily detected in PTr2 cells by IF, FCM, RT-qPCR, TEM and IEM techniques. RT-qPCR and FCM results of a time course of infection of PTr2 cells indicated PRRSV-2 load decreased over time after initial infection up to 72 h. PRRSV-2 infection altered PTr2 cell cycle with a selective increase of cells within the G2/M phase and also induced apoptosis. TEM and IEM demonstrated PRRSV-2 within and on the surface of PTr2 cells and PRRSV-2 virions released from PTr2 cells infected naïve MARC-145 cells inducing cytopathic effects. DISCUSSION: Trophoblast cells are susceptible to PRRSV-2 infection and release live virions capable of inducing cytopathic effects in naïve cells. This suggests a possible mechanism by which PRRSV-2 can breach the MFI resulting in fetal infection and death.

Spatiotemporal immunofluorescent evaluation of porcine reproductive and respiratory syndrome virus transmission across the maternal-fetal interface.

Porcine reproductive and respiratory syndrome virus (PRRSV) infection causes severe reproductive failure characterized by high fetal morbidity and mortality leading to substantial economic losses to the swine industry. Evaluation of spatiotemporal transmission of PRRSV at the maternal-fetal interface (MFI) is critical for understanding fetal infection. Localization of PRRSV-2 strain NVSL 97-7895 at different regions of the MFI in 20 pregnant gilts at 2, 5, 8, 12 and 14 days post-inoculation (dpi) were analyzed by immunofluorescence (IF). Samples of MFI were collected from 15 inoculated and 5 control gilts and transplacental PRRSV transmission assessed in randomly selected fetuses from each litter. Localization of NVSL 97-7895 antigen immunoreactivity in the MFI was focused in three major areas: endometrial connective tissues (ENDO), the feto-maternal junction (FMJ) and fetal placenta (PLC). NVSL 97-7895 was detected at the FMJ by 2 dpi. At 2, 5 and 8 dpi, NVSL 97-7895 was localized within the ENDO and FMJ, whereas at 12 and 14 dpi, it was mainly localized in the PLC. Using a novel IF strategy for counting and size sorting NVSL 97-7895 viral antigen in situ, results of this study indicate that non-cell-associated mechanisms are involved in PRRSV transmission across the MFI.

In vitro characterization of influenza A virus attachment in the upper and lower respiratory tracts of pigs.

The binding of influenza A viruses to epithelial cells in the respiratory tract of mammals is a key step in the infection process. Therefore, direct assessment of virus-host cell interaction using virus histochemistry (VH) will enhance our understanding of the pathogenesis of these new viruses. For this study, the authors selected viruses that represented the 4 main genetic clusters of North American swine H1 (SwH1) viruses, along with A/California/04/2009 H1N1 and a vaccine strain for the positive controls, and the virus label, fluorescein isothiocyanate (FITC), for the negative control. A group of 5 viruses containing a 2-amino acid insertion adjacent to the binding site of the hemagglutinin protein and their presumed ancestral viruses were also examined for changes in binding patterns. Viruses were bound to formalin-fixed paraffin-embedded, 6-week-old (6w) and adult pig tissues. Qualitative VH scores per respiratory zone ranged from + to +++, with bronchioles having the highest and most consistent scores, regardless of animal age. For the 6w bronchioles, a quantitative VH score was calculated using digital images of 5 bronchioles per tissue section using image analysis software. Significant differences in attachment were found among the SwH1 viruses (P < .0001) and among the ancestral and insertion viruses (P < .0001). These results provide new insights on virus binding to porcine respiratory epithelial cells and the usefulness of morphometric scores. The results also highlight limitations of in vitro techniques, including VH for predicting virulence and host range.