The effect of infection order of porcine circovirus type 2 and porcine reproductive and respiratory syndrome virus on dually infected swine alveolar macrophages

Abstract

Background: Concurrent infection with porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) is known as one of the major causes for porcine respiratory disease complex (PRDC). Dual infection with PCV2 and PRRSV is consistently to have more severe clinical presentations and pulmonary lesions than infection with PCV2 alone or PRRSV alone. However, it is not known if dual infections with PCV2 and PRRSV in different infection order may lead to different clinical symptoms in the host. To mimic the possible field conditions, swine alveolar macrophages (AMs) were inoculated with PCV2 and PRRSV in vitro simultaneously or with one virus 18 h earlier than the other. The cell viability, cytopathic effects, antigen-containing rates, phagocytotic and microbial killing capabilities, cytokine profiles (IL-8, TNF-α, and IFN-α) and FasL transcripts were determined, analyzed, and compared to prove the hypothesis.

Results: A marked reduction in PRRSV antigen-containing rate, cytopathic effect, and TNF-α expression level was revealed in AMs inoculated with PCV2 and PRRSV simultaneously and in AMs inoculated with PCV2 first then PRRSV 18 h later, but not in AMs inoculated with PRRSV first then PCV2 18 h later. Transient decrease in phagocytosis but constant reduction in microbicidal capability in AMs in the group inoculated with PCV2 alone and constant decrease in phagocytosis and microbicidal capability in AMs in all PRRSV-inoculated groups were noted. The levels of IL-8, TNF-α, IFN-α, and FasL transcripts in AMs in all groups with dual inoculation of PCV2 and PRRSV were significantly increased regardless of the infection orders as compared with infection by PCV2 alone or PRRSV alone.

Conclusions: Swine AMs infected with PCV2 first then PRRSV later or infected with PCV2 and PRRSV simultaneously displayed marked reduction in PRRSV antigen-containing rate, cytopathic effect, and TNF-α expression level. The different inoculation orders of PCV2 and PRRSV in AMs leading to different results in viral antigen positivity, cytopathology, and cytokine profile may explain, at least partially, the underlying mechanism of the enhanced pulmonary lesions in PRDC exerted by dual infection with PCV2 and PRRSV and the variable clinical manifestations of PRDC-affected pigs in the field.

Figure 1

Changes in PCV2 and PRRSV antigen-positive rate in swine alveolar macrophages (AMs). Changes in (A) PCV2 and (B) PRRSV antigen-positive rate in PCV2- and/or PRRSV-inoculated swine alveolar macrophages (AMs) were determined by indirect immunofluorescence assay. Data are expressed as percentage and shown as mean ± SD of three independent experiments. Solid diamond: inoculation with PCV2 alone (PCV2); open diamond: inoculation with PRRSV alone (PRRSV); solid square: inoculation with PCV2 and PRRSV simultaneously (PCV2-PRRSV); solid triangle: inoculation with PCV2 for 18 h first then PRRSV later (PCV2/PRRSV); open triangle: inoculation with PRRSV for 18 h first then PCV2 later (PRRSV/PCV2). *** ^{(P < 0.001)} The values are significantly different from PRRSV or PRRSV/PCV2 at the same h post PRRSV inoculation.

Figure 2

Changes in survival rate and apoptotic rate of swine alveolar macrophages (AMs). Changes in (A) survival rate and (B) apoptotic rate in PCV2- and/or PRRSV-inoculated swine alveolar macrophages (AMs) were determined by trypan blue dye exclusion assay and TUNEL assay, respectively. Data are expressed as the level different from that of the mock-inoculated AMs (Mock) and shown as mean ± SD of three independent experiments. PCV2: AMs inoculated with PCV2 alone; PRRSV: AMs inoculated with PRRSV alone; PCV2/PRRSV: AMs inoculated with PCV2 first then inoculated with PRRSV 18 h later; PRRSV/PCV2: AMs inoculated with PRRSV first then inoculated with PCV2 18 h later; PCV2-PRRSV: AMs co-inoculated with PCV2 and PRRSV simultaneously. ** ^{(P < 0.01),} *** ^{(P < 0.001)}The difference between the two treatment groups at the same h post inoculation (HPI) with the first virus is statistically significant. ^aValues are significantly different from the Mock at the same h post inoculation (HPI) with the first virus.

Figure 3

Changes in phagocytotic and microbial killing capabilities of swine alveolar macrophages (AMs). Changes in (A) phagocytotic and (B) microbial killing capabilities in PCV2- and/or PRRSV-inoculated swine alveolar macrophages (AMs) were determined by using Candida albicans as the target. Following 60 min of incubation and staining with acridine orange, the percentages of viable AMs with engulfed yeasts and killed yeasts were determined. Data are expressed as the level different from that of the mock-inoculated AMs (Mock) and shown as mean ± SD of three independent experiments. PCV2: AMs inoculated with PCV2 alone; PRRSV: AMs inoculated with PRRSV alone; PCV2/PRRSV: AMs inoculated with PCV2 first then inoculated with PRRSV 18 h later; PRRSV/PCV2: AMs inoculated with PRRSV first then inoculated with PCV2 18 h later; PCV2-PRRSV: AMs co-inoculated with PCV2 and PRRSV simultaneously. * ^{(P < 0.05),} ** ^{(P < 0.01),} *** ^{(P < 0.001)}The difference between the two treatment groups at the same h post inoculation (HPI) with the first virus is statistically significant. ^aValues are significantly different from the Mock at the same h post inoculation (HPI) with the first virus.

Figure 4

Changes in the levels of IL-8, TNF-α, and IFN-α production in the supernatants of swine alveolar macrophages (AMs). Changes in the levels of IL-8 (A), TNF-α (B), and IFN-α (C) produced in PCV2- and/or PRRSV-inoculated swine alveolar macrophages (AMs) were expressed as the level different from that of mock-inoculated AMs (Mock) and shown as mean ± SD of three independent experiments. PCV2: AMs inoculated with PCV2 alone; PRRSV: AMs inoculated with PRRSV alone; PCV2/PRRSV: AMs inoculated with PCV2 first then inoculated with PRRSV 18 h later; PRRSV/PCV2: AMs inoculated with PRRSV first then inoculated with PCV2 18 h later; PCV2-PRRSV: AMs co-inoculated with PCV2 and PRRSV simultaneously. *Significantly different (P < 0.05) from the Mock at the same h post inoculation (HPI) with the first virus. ^{a, b, c, d, e}Values with different labels at the same HPI differ significantly (P < 0.05).

Figure 5

Expression levels of FasL mRNA in PCV2- and/or PRRSV-inoculated swine alveolar macrophages (AMs). The expression levels of FasL mRNA in all cultures were analyzed at 42 h after inoculation with the first virus by RT-PCR and electrophoresis. The sizes of FasL and GAPDH housekeeping gene are 366 and 96 b.p., respectively (A); values are further normalized using the housekeeping gene GAPDH and expressed as the relative intensity at the level different from that of mock-inoculated AMs (Mock) and shown as mean ± SD of three independent experiments (B). Mock: AMs inoculated with an equal volume of RPMI-C; PCV2: AMs inoculated with PCV2 alone; PRRSV: AMs inoculated with PRRSV alone; PCV2/PRRSV: AMs inoculated with PCV2 first then inoculated with PRRSV 18 h later; PRRSV/PCV2: AMs inoculated with PRRSV first then inoculated with PCV2 18 h later; PCV2-PRRSV: AMs co-inoculated with PCV2 and PRRSV simultaneously. *Significantly different (P < 0.05) from the Mock. ^{a, b, c}Values with different labels differ significantly (P < 0.05).