In vitro and ex vivo analyses of co-infections with swine influenza and porcine reproductive and respiratory syndrome viruses

Abstract

Viral respiratory diseases remain problematic in swine. Among viruses, porcine reproductive and respiratory syndrome virus (PRRSV) and swine influenza virus (SIV), alone or in combination, are the two main known contributors to lung infectious diseases. Previous studies demonstrated that experimental dual infections of pigs with PRRSV followed by SIV can cause more severe disease than the single viral infections. However, our understanding of the impact of one virus on the other at the molecular level is still extremely limited. Thus, the aim of the current study was to determine the influence of dual infections, compared to single infections, in porcine alveolar macrophages (PAMs) and precision cut lung slices (PCLS). PAMs were isolated and PCLS were acquired from the lungs of healthy 8-week-old pigs. Then, PRRSV (ATCC VR-2385) and a local SIV strain of H1N1 subtype (A/Sw/Saskatchewan/18789/02) were applied simultaneously or with 3h apart on PAMs and PCLS for a total of 18 h. Immuno-staining for both viruses and beta-tubulin, real-time quantitative PCR and ELISA assays targeting various genes (pathogen recognition receptors, interferons (IFN) type I, cytokines, and IFN-inducible genes) and proteins were performed to analyze the cell and the tissue responses. Interference caused by the first virus on replication of the second virus was observed, though limited. On the host side, a synergistic effect between PRRSV and SIV co-infections was observed for some transcripts such as TLR3, RIG-I, and IFNβ in PCLS. The PRRSV infection 3h prior to SIV infection reduced the response to SIV while the SIV infection prior to PRRSV infection had limited impact on the second infection. This study is the first to show an impact of PRRSV/SIV co-infection and superinfections in the cellular and tissue immune response at the molecular level. It opens the door to further research in this exciting and intriguing field.

Keywords: Co-infection; Interference; PRRSV; Pig; SIV; Superinfection.

Fig. 1

Viability of PCLS evaluated by bronchoconstriction 96 h after the PCLS preparation. Untreated slice (a) was incubated with 10⁻⁴ M methacholine (b) to induce bronchoconstriction. Removal of the drug resulted in a reverse effect (c). Viability was tested at 24 h and 96 h after the PCLS preparation. Representative of two independent experiments.

Fig. 2

Infection of PCLS by SIV and PRRSV characterized by immunostaining. PCLS were infected by SIV (A), PRRSV (B) or both viruses (C). Cryosections were prepared after 18 h of infection and image data was collected using a laser-scanning confocal microscope. Infected cells were stained with an anti-nucleoprotein polyclonal antibody (green in A and red in C) for the detection of SIV (green in A and red in C) and with an anti-nucleocapsid monoclonal antibody to detect PRRSV (green in B and C). Ciliated cells were stained using an anti-beta-tubulin monoclonal antibody (red in A and B). White arrows indicated infected cells in each panel, scale bar = 20 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Fig. 3

PRRSV cellular targets in alveolar tissue. PCLS was infected by PRRSV and SIV. Cryosections were prepared after 18 h of infection and used for detection of infected cells in the alveoli. Infected cells were stained with an anti-nucleocapsid monoclonal antibody for the detection of PRRSV (green). Ciliated cells were stained using an anti-beta-tubulin monoclonal antibody (red). Arrows indicate infected cells (presumably type 1 cell and macrophage, horizontal arrow), scale bar = 20 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Fig. 4

Viral replication – relative expression of viral transcripts (nucleoprotein-PRRSV and M protein-SIV) after 18 h of infection of PCLS and PAMs. For the PCLS, n = 6 slices and median value in one representative pig out of four and for PAMs, n = 6 pigs and median value. Dot plots within each graph with no common superscripts are significantly different (P < 0.05).

Fig. 5

Viral recognition – relative expression of viral recognition transcripts after 18 h of infection of PCLS (A) and PAMs (B). For the PCLS, n = 6 slices and median value in one representative pig out of four and for PAMs, n = 6 pigs and median value. Dot plots within each graph with no common superscripts are significantly different (P < 0.05).

Fig. 6

Interferons – relative expression of interferon transcripts after 18 h of infection of PCLS and PAMs. For the PCLS, n = 6 slices and median value in one representative pig out of four and for PAMs, n = 6 pigs and median value. Dot plots within each graph with no common superscripts are significantly different (P < 0.05).

Fig. 7

Response to interferons – relative expression of interferon induced gene transcripts after 18 h of infection of PCLS and PAMs. For the PCLS, n = 6 slices and median value in one representative pig out of four and for PAMs, n = 6 pigs and median value. Dot plots within each graph with no common superscripts are significantly different (P < 0.05).