Porcine reproductive and respiratory syndrome virus modifies innate immunity and alters disease outcome in pigs subsequently infected with porcine respiratory coronavirus: implications for respiratory viral co-infections - PubMed (original) (raw)

Porcine reproductive and respiratory syndrome virus modifies innate immunity and alters disease outcome in pigs subsequently infected with porcine respiratory coronavirus: implications for respiratory viral co-infections

Kwonil Jung et al. J Gen Virol. 2009 Nov.

Abstract

The innate immune response is critical for host defence against respiratory coronaviruses (CoVs). This study demonstrated that an ongoing respiratory virus infection compromises innate immune responses and affects the pathogenesis of a respiratory CoV co-infection. An innate immunosuppressive respiratory virus infection was established by infecting weaned pigs with porcine reproductive and respiratory syndrome virus (PRRSV); 10 days later, the pigs were exposed to porcine respiratory coronavirus (PRCV). The PRRSV/PRCV dual-infected pigs had reduced weight gains, a higher incidence of fever and more severe pneumonia compared with either single infection. Significant suppression of innate immune responses [reduced alpha interferon (IFN-alpha) levels in the lungs and reduced blood natural killer cell cytotoxicity] by the ongoing PRRSV infection was observed in dual-infected pigs, which coincided with exacerbated pneumonia during early PRCV infection. The subsequent PRCV infection led to enhanced PRRSV replication in the lungs and a trend towards increased serum T-helper type 1 (Th1) (IFN-gamma) but decreased Th2 [interleukin (IL)-4] responses, further exacerbating PRRSV pneumonia. Following PRCV infection, more severe PRRSV-related pulmonary alveolar macrophage (PAM) apoptosis occurred, as determined by an in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling assay, suggesting increased PRRSV replication in PAMs. Collectively, these observations suggest interactive effects between PRCV and PRRSV via early innate (IFN-alpha) and later adaptive Th1 (IFN-gamma) and Th2 (IL-4) immune responses. These findings imply that an existing immunomodulating respiratory viral co-infection may be a contributing factor to more severe pneumonia in respiratory CoV disease. This study provides new insights into host-pathogen interactions related to co-infection by CoVs and other respiratory viruses.

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Figures

Fig. 1.

Fig. 1.

Ongoing PRRSV and subsequent PRCV co-infection exacerbate pneumonia compared with pigs infected with PRRSV or PRCV alone. (a) Gross evidence of pneumonia at PRCV PIDs 2 and 8. (b–j) Lung sections from: (b) a PRRSV/PRCV-infected pig at PRCV PID 2, showing severe bronchointerstitial pneumonia; (c) a PRRSV-infected pig at PRCV PID 2, showing moderate bronchointerstitial pneumonia; (d) a PRCV only-infected pig at PRCV PID 2, showing moderate interstitial pneumonia; (e, h) a PRRSV/PRCV-infected pig at PRCV PID 8, showing severe bronchointerstitial pneumonia characterized by accumulation of necrotic cells (N) in alveolar spaces and type 2 pneumocyte (T2) hyperplasia and alveolar macrophage infiltration (A) in alveolar septa, accompanying lymphohistiocytic perivascular cuffing (C); sometimes, type 1 pneumocytes (T1) formed the alveolar epithelium; (f, i) a PRRSV-infected pig at PRCV PID 8, showing moderate to severe bronchointerstitial pneumonia characterized by accumulation of necrotic cells (N) and massive infiltration of alveolar macrophages (A) that had apoptotic bodies (Apb) in their nuclei; (g, j) a PRCV only-infected pig at PRCV PID 8, showing severe interstitial pneumonia in which type 2 pneumoytes have infiltrated the alveolar septa. Magnification, ×100 (b–g); ×400 (h–j).

Fig. 2.

Fig. 2.

High Th1 (IFN-γ) and low Th2 (IL-4) serum cytokine responses coincide with the prolonged pneumonia observed in dual-infected pigs. The pigs (_n_=6–8 at PRCV PIDs −2 to 14 and _n_=5 at PID 21 for PRRSV/PRCV and PRCV alone; _n_=5–6 at each PID for PRRSV alone; _n_=6–7 at each PID for mock infections) were necropsied and assessed for serum cytokine ELISA. (a) Gross lung lesion score. Gross and histological lung lesions were given an estimated score based on the percentage of macroscopic consolidation in all lobes and the distribution and severity of histopathology, respectively. (b) Histological lung lesion score. (c) Th1 (IFN-γ) cytokine serum levels. (d) Th2 (IL-4) cytokine serum levels. Each bar represents the mean±

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. *P<0.05; **P<0.01 (statistically significant differences between the dually and singly infected pigs by the Kruskal–Wallis test).

Fig. 3.

Fig. 3.

Suppression of innate immune responses (reduced IFN-α levels in lung and reduced blood NK cell cytotoxicity) by ongoing PRRSV infection coincides with exacerbated pneumonia during early PRCV infection. (a) IFN-α in the lungs. Lung lysates were prepared from pigs at each PID with the numbers of pigs indicated in the legend for Fig. 2 and tested for IFN-α levels by ELISA. (b) NK cell cytotoxicity (%) was measured using PBMCs (effectors) harvested from pigs at each PID against target cells (K-562 or Yac-1). Effectors and targets at the indicated ratio (100 : 1) were co-cultured and the supernatants harvested after 24 h. The amount of LDH released was measured by using LDH substrate and measuring absorbance at 490 nm. Each bar represents the mean percentage of NK-specific lysis of targets from two or three pigs±

sem

. (c, d) PRCV replication in the lungs. Paraffin-embedded lung tissues were evaluated by IHC for PRCV antigen detection (c). Lung homogenates were also tested by qRT-PCR for viral RNA quantification (d). Each data point represents the mean±

sem

. *P<0.05 (statistically significant difference between the dual-infected and the singly or mock-infected pigs by the Kruskal–Wallis test). (e, f) Lung section from a PRCV only-infected pig at PRCV PID 4, showing the large number of PRCV-positive cells in the lung by IHC. PRCV antigens were identified in bronchiolar epithelial cells (f; arrowheads) and type 2 pneumocytes (arrow). (g, h) Lung section from a PRRSV/PRCV-infected pig at PRCV PID 4, showing moderate numbers of PRCV-positive cells (h; arrow) in the alveolar septa by IHC. (i) Lung section from a PRRSV/PRCV-infected pig at PRCV PID 8, showing large numbers of PRRSV-positive cells in the alveolar septa and spaces by IHC. PRRSV antigens were identified in alveolar macrophages (arrow). (j) Lung section from a PRRSV-infected pig at PRCV PID 8, showing small numbers of PRRSV-positive cells (arrow) by IHC. Magnification: ×200 (e, g, i, j); ×400 (f, h).

Fig. 4.

Fig. 4.

Subsequent PRCV infection promotes PRRSV replication in the lungs and induction of severe PRRSV-related apoptotic lesions at PRCV PIDs 4–10. (a, b) PRRSV replication in the lungs. Each data point represents the mean±

sem

. *P<0.05 (statistically significant differences between dual-infected pigs and pigs singly infected by either virus by the Kruskal–Wallis test. (c) Paraffin-embedded lung tissues were evaluated by an in situ TUNEL assay (black staining) for detection of apoptosis. Magnification: ×50. Cells were counterstained with methyl green. +, A few positive cells; ++, moderate numbers of positive cells; +++, many positive cells.

Fig. 5.

Fig. 5.

PRCV- and PRRSV-specific serum VN and ELISA antibody titres in pigs co-infected with PRRSV and PRCV. Serum was collected at each PID from the numbers of pigs indicated in the legend of Fig. 2. (a) Serum VN antibody titres against PRCV were evaluated by a plaque-reduction test. Antibody titres were expressed as the reciprocal of the specimen dilution that resulted in an 80 % reduction in the number of plaques. (b) PRRSV serum antibodies were identified by a commercial PRRSV ELISA (Idexx Laboratories, ME) and evaluated based on progression of the S : P ratios. Each data point represents the mean±

sem

.

Fig. 6.

Fig. 6.

PRCV nasal shedding titres (a) and PRCV-specific IgA antibodies (b) in BAL of pigs co-infected with PRRSV and PRCV. (a) Nasal swabs were collected from each pig (_n_=39 at PRCV PID 2, _n_=31 at PID 4, _n_=20 at PID 6, _n_=15 at PID 8, _n_=11 at PID 10 and _n_=5–6 at PIDs 12–21 for PRRSV/PRCV-infected and PRCV only-infected pigs) at PRRSV PIDs 0–31 and tested by a cell culture immunofluorescence assay. The numbers of PRCV only-infected cells were expressed as f.f.u. ml−1. (b) BAL fluids were prepared at each PID from the numbers of pigs indicated in the legend of Fig. 2. PRCV-specific IgA antibody titres in BAL fluids were tested by ELISA. Each data point represents the S : P ratio (mean±

sem

). *P<0.05 (statistically significant difference between the dual-infected and PRCV only-infected pigs by the Kruskal–Wallis test).

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