Interleukin-1β expression by a recombinant porcine reproductive and respiratory syndrome virus

Abstract

The cytokine interleukin-1 beta (IL-1β) is a potent inflammatory mediator in response to infection, and can be used as an immunological adjuvant. In this study, we constructed a recombinant porcine reproductive and respiratory syndrome virus (vP129/swIL1β) expressing swine IL-1β from the separate subgenomic mRNA inserted between the ORF1b and ORF2 genome region. MARC-145 cells infected with vP129/swIL1β secreted 1947 pg of IL-1β per 2 × 10(5)cells at 36 h post-infection. In vitro growth kinetics analysis in MARC-145 cells showed that the vP129/swIL1β virus had a similar replication rate as that of parental virus. We further performed in vivo characterization of the vP129/swIL1β virus in a nursery pig disease model. The vP129/swIL1β infected pigs did not show visible clinical signs, while respiratory distress and lethargy were evident in pigs infected with the parental virus. The expression of various cytokines from peripheral blood mononuclear cells measured by fluorescent microsphere immunoassay showed that IL-1β, IL-4 and IFN-γ expression levels were up-regulated in pigs infected with vP129/swIL1β at 7 and 14 days post-infection. However, no detectable level of IL-1β was found in serum samples from pigs infected with either vP129/swIL1β or parental virus. In summary, this study demonstrated a recombinant PRRSV as a useful tool to study the role of different cytokines in disease progression and immune responses, which represents a new strategy for future therapeutic application and vaccine development.

Fig. 1

Expression of swIL1β by vP129/swIL1β recombinant virus. (A) Construction of the plasmid, pCMV-S-P129-1bswIL1β. The GFP gene in pCMV-S-P129-1bGFP2 (Kermit) backbone was replaced by swine IL-1β gene. The IL-1β is expressed as an independent subgenomic RNA, which was derived from the leader/junction (L/J) sequence of ORF2. The ORF6 L/J was inserted downstream of the IL-1β gene to drive the expression of ORF2; (B) Western blot analysis of the detection of swIL1β in the supernatant fraction (sup) or cell lysates (cell lys) of MARC-145 cells infected with vP129/swIL1β or parental P129 virus. Cells were infected with the MOI of 1 and incubated for 24 h before sample preparation for Western blot analysis, and (C) kinetics of accumulation of swine IL-1β in culture supernatant of MARC-145 cells infected with vP129/swIL1β or parental virus. MARC-145 cells were infected in parallel at MOI of 0.5, and culture supernatant was collected at indicated time points. The swine IL-1β expression levels were determined by Luminex fluorescent microsphere immunoassay (FMIA).

Fig. 2

In vitro growth characterization of vP129/swIL1β recombinant virus. (A) Growth kinetics comparison between vP129/swIL1β and parental virus in MARC-145 cells. MARC-145 cells were infected in parallel at MOI of 0.1 with the passage three of chimeric or parental virus. At 6, 12, 24, 36, 48, 60, and 72 h post-infection, cells were harvested, and the virus titers were determined by IFA on MARC-145 cells. The results were mean values from three replications of the experiment, and viral titers were expressed as fluorescence focus units per milliliter (FFU/ml), and (B) Plaque morphology of vP129/swIL1β and parental virus. Confluent cell culture monolayers were infected with viruses at a MOI of 0.1. After 2 h infection, cell culture supernatant was removed and an agar overlay was applied. Plaques were detected after five days incubation at 37 °C and stained by 0.1% crystal violet.

Fig. 3

Viral load in pigs infected with vP129/swIL1β or parental virus. Viral load was quantified by real-time RT-PCR, and the result was interpreted as RNA copy numbers per ml. (A) Viral load in serum samples, and (B) Viral load in tissue samples. Different letters (“a” and “b”) represent mean viral load of different groups differs significantly (P < 0.05).

Fig. 4

Comparison of cytokine expression levels in activated PBMCs from pigs infected by vP129/swIL1β with that of parental virus. PBMCs were harvested at 3, 7 and 14 dpi and stimulated in culture with PHA or medium. Cell culture supernatants and pellets were harvested at 24 h post-stimulation. Cytokine protein expression was determined by Luminex fluorescent microsphere immunoassay. Each data point was interpolated using a five-parameter logistic regression curve and displayed as a mean fluorescent unit from three animals with the standard deviation. Different letters (“a”, “b” and “c”) represent mean cytokine level of different groups differs significantly (P < 0.05).

Fig. 5

Antibody response in pigs infected with vP129/swIL1β or parental virus. (A) PRRSV-specific serum antibodies measured by IDEXX HerdChek^® PRRSV ELISA 2XR kit. S/P ratios of greater than 0.4 are considered positive. (B) Serum neutralizing antibody response determined by fluorescent focus neutralization assay. Results were interpreted as a 90% reduction of the viral infection, and the neutralizing antibody titers were presented as mean value (n = 6) and expressed on a log₂ scale. The parental virus was used for the viral neutralizing assay. Different letters (“a”, “b” and “c”) represent mean neutralizing antibody titer of different groups differs significantly (P < 0.05).