Reduction of porcine reproductive and respiratory syndrome virus (PRRSV) infection in swine alveolar macrophages by porcine circovirus 2 (PCV2)-induced interferon-alpha

Abstract

Two common viral pathogens of swine, namely, porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV), were investigated in regard to their effects on monolayer cultures of swine alveolar macrophages (AMs). The purpose was to identify selected cellular changes and responses potentially associated with the clinical reactions of pigs infected with either or both of these viruses. Measurements included the (1) absolute and relative numbers of infected, viable, and apoptotic cells; (2) distribution of viral antigens; (3) levels of interferon-alpha (IFN-alpha) and tumor necrosis factor-alpha (TNF-alpha) produced and their association with the extent of virus-induced cytopathology. Four groups of AMs were studied, including mock-infected, PCV2 alone-infected (PCV2-A), PRRSV alone-infected (PRRSV-A), and PCV2 and PRRSV dually infected (PCV2/PRRSV) groups. The AMs of PCV2-A group had high antigen-containing rate without cell death. There was a marked increase in cell death and apoptosis in PRRSV-A group. However, a lower PRRSV-induced infectious rate, cell death, and apoptosis were seen in PCV2/PRRSV group. High levels of IFN-alpha production were detected in PCV2-infected groups, but not in mock-infected and PRRSV-A groups. The PRRSV-induced cytopathic effect (CPE) on MARC-145 cells or swine AMs was markedly reduced by pre-incubation of the cells with UV-treated or non-UV-treated supernatants of PCV2-infected AMs. In addition, the reduction in CPE was abolished when the supernatants of PCV2-infected AMs were pre-treated with a mouse anti-recombinant porcine IFN-alpha antibody. The results suggest that swine AMs were an important reservoir of PCV2; PCV2 infection reduced PRRSV infection and PRRSV-associated CPE in PCV2/PRRSV AMs; the reduction of PRRSV infection in AMs was mediated by IFN-alpha generated by PCV2 infection. The reduced PRRSV-associated CPE in AMs and increased pro-inflammatory cytokine production may lead to a more severe pneumonic lesion in those dually infected pigs.

Fig. 1

The results of immunofluorescence antibody staining for mock-infected and porcine circovirus 2 (PCV2)-infected swine alveolar macrophages (AMs). (A) No PCV2 antigen is present in mock-infected group; ×450. (B) Approximately 97% of the AMs in PCV2 alone-infected group have positive signal in the cytoplasm at 18 h post-infection (HPI); ×450. Inset: a pinpoint to small granular, intracytoplasmic bright red fluorescence (Texas-red) is seen in PCV2-infected AMs; ×1250. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

The results of immunofluorescence antibody staining for porcine reproductive and respiratory syndrome virus (PRRSV)-infected swine alveolar macrophages (AMs). A diffuse, intracytoplasmic bright green fluorescence (fluorescein isothiocyanate, FITC) is seen in PRRSV-infected AMs. (A) Only approximately 9.3% of AMs have positive signal in PRRSV alone-infected group at 18 h post-infection (HPI); ×450. Inset: a non-PRRSV-infected but PRRSV-positive apoptotic body-containing AM. Note the scattered intracytoplasmic granular to globular fluorescence; ×1250. (B) In porcine circovirus 2 and PRRSV dually infected group, the rate of PRRSV-positive AMs was only 1.2% by 108 HPI; ×450. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Changes in cell viability of swine alveolar macrophages (AMs) over time following infection with porcine circovirus 2 (PCV2) and/or porcine reproductive and respiratory syndrome virus (PRRSV) as determined by trypan blue dye exclusion assay. Data are expressed as percentage and shown as mean ± S.D. of three independent experiments. Con: mock-infected group; PCV2-A: PCV2 alone-infected group; PRRSV-A: PRRSV alone-infected group; PCV2/PRRSV: PCV2 and PRRSV dually infected group. The differences between PCV2 and/or PRRSV-infected and mock-infected groups at the same hour post-infection (HPI) are significant (^*P < 0.05). The two values labeled at the same HPI differ significantly (^☆P < 0.05).

Fig. 4

Changes in apoptotic rate of swine alveolar macrophages (AMs) over time following infection with porcine circovirus 2 (PCV2) and/or porcine reproductive and respiratory syndrome virus (PRRSV) determined by TUNEL assay. Data are expressed as percentage and shown as mean ± S.D. of three independent experiments. Con: mock-infected group; PCV2-A: PCV2 alone-infected group; PRRSV-A: PRRSV alone-infected group; PCV2/PRRSV: PCV2 and PRRSV dually infected group. The differences between PCV2 and/or PRRSV-infected and mock-infected groups at the same hour post-infection (HPI) are significant (^*P < 0.05). The two values labeled at the same HPI differ significantly (^☆P < 0.05).

Fig. 5

Protective effect of culture supernatants from mock-infected and porcine circovirus 2 (PCV2) and/or porcine reproductive and respiratory syndrome virus (PRRSV)-infected swine alveolar macrophages (AMs) collected at various time intervals after infection determined by cytopathic effect (CPE) reduction bioassay using Madin–Darby bovine kidney cells and vesicular stomatitis virus (VSV). Crystal violet is used to stain the viable cells. Complete (A) or almost complete (B) protection from VSV-induced CPE is found by adding the supernatant of PCV2 alone-infected or PCV2 and PRRSV dually infected swine AMs collected at 36 h post-infection (HPI), respectively; ×115. No protection from VSV-induced CPE is found by adding the supernatant of PRRSV alone-infected AMs at 36 HPI (C); ×115.

Fig. 6

Changes in the levels of IFN-α in the culture supernatants over time following infection with porcine circovirus 2 (PCV2) and/or porcine reproductive and respiratory syndrome virus (PRRSV) determined by cytopathic effect (CPE) reduction bioassay using Madin–Darby bovine kidney (MDBK) cells and vesicular stomatitis virus (VSV). One unit of IFN-α is defined as the reciprocal of the dilution of the supernatant producing 50% inhibition of CPE. Data are expressed as U/ml and shown as mean ± S.D. of three independent experiments. Con: mock-infected group; PCV2-A: PCV2 alone-infected group; PRRSV-A: PRRSV alone-infected group; PCV2/PRRSV: PCV2 and PRRSV dually infected group. The differences between PCV2 and/or PRRSV-infected and mock-infected groups at the same hour post-infection (HPI) are significant (^*P < 0.05).

Fig. 7

Changes in the level of TNF-α in the culture supernatants of swine alveolar macrophages (AMs) over time following infection with porcine circovirus 2 (PCV2) and/or porcine reproductive and respiratory syndrome virus (PRRSV) determined by an ELISA kit. Data are expressed as pg/ml and shown as mean ± S.D. of three independent experiments. Con: mock-infected group; PCV2-A: PCV2 alone-infected group; PRRSV-A: PRRSV alone-infected group; PCV2/PRRSV: PCV2 and PRRSV dually infected group. The differences between PCV2 and/or PRRSV-infected and mock-infected groups at the same hour post-infection (HPI) are significant (^*P < 0.05). The two values labeled at the same HPI differ significantly (^☆P < 0.05).

Fig. 8

Protection of MARC-145 cells from porcine reproductive and respiratory syndrome virus (PRRSV) infection by culture supernatants of porcine circovirus 2 (PCV2)-infected swine alveolar macrophages (AMs). Crystal violet is used to stain the survival cells; ×115. Complete (A) or almost complete (B) protection from PRRSV-induced CPE is found by adding UV-treated or non-UV-treated supernatant of PCV2-infected AMs collected at 54 HPI, respectively; ×115. No protection from PRRSV-induced CPE is found by adding the AM supernatant pre-mixed with a mouse anti-recombinant porcine IFN-α antibody (C); ×115.