Nitric oxide is elicited and inhibits viral replication in pigs infected with porcine respiratory coronavirus but not porcine reproductive and respiratory syndrome virus

Abstract

There is little information on the role of nitric oxide (NO) in innate immunity to respiratory coronavirus (CoV) infections. We examined NO levels by Greiss assay in bronchoalveolar lavage (BAL) of pigs infected with either porcine respiratory coronavirus (PRCV) or porcine reproductive and respiratory syndrome virus (PRRSV), a member of Nidovirales, like CoV. The antiviral effects of NO on these two viruses were tested in an in vitro system using a NO donor, S-nitroso-N-acetylpenicillamine (SNAP). We detected a large increase in NO levels in BAL fluids of PRCV-infected pigs, but not in PRRSV-infected pigs. Pulmonary epithelial cell necrosis induced by PRCV coincided with increased NO. Moreover, NO levels in cell culture medium of PRRSV-infected alveolar macrophages (AMs) did not differ from that of mock-infected AMs. Antiviral assays showed that NO significantly inhibited PRCV replication in swine testicular (ST) cells, whereas PRRSV was not susceptible to NO based on the conditions tested. Our study suggests that unlike PRRSV which induces apoptosis in AMs, respiratory CoVs such as PRCV that infect pulmonary epithelial cells and cause cytolysis, induce NO production in the respiratory tract. Thus, NO may play a role in innate immunity to respiratory CoV infections by inhibiting viral replication.

Fig. 1

PRCV, but not PRRSV, induced increased •NO levels in BAL during the early and middle stages of infection (PRCV PID 2–10). (A and B) BAL •NO levels in different independent animal trials. BAL samples were collected from each pig (n = 1 at all PIDs for mock and PRRSV single-infected pigs, n = 1–2 at each PID for PRRSV/PRCV dual- and PRCV single-infected pigs). All data are expressed as the means ± SDM. (C) Mean BAL •NO levels in four independent animal trials. BAL samples were collected from each pig (n = 4 at all PIDs for mock and PRRSV single-infected pigs, n = 4–6 at each PID for PRRSV/PRCV dual- and PRCV single-infected pigs). All data are expressed as the means ± SEM. Pigs were first inoculated with PRRSV or mock and, 10 days later, inoculated with PRCV or mock. Total •NO (nitrite and nitrate) in BAL fluids were measured by the Griess method. All assays were performed in duplicate.

Fig. 2

•NO significantly inhibited PRCV replication in ST cells, whereas PRRSV was not susceptible to •NO. (A and C) •NO production in ST and MARC145 cells, respectively. ST and MARC145 cells were treated with SNAP at different concentrations (0, 50, 100, 200, 400, and 800 μM), and cell supernatants were harvested at PTH 24 and 48 for the Griess assay. (B and D) The antiviral effects of •NO on PRCV replication in ST cells and PRRSV replication in MARC145 cells, respectively. The SNAP-treated, virus-infected and SNAP-untreated, virus-infected cells were infected with PRCV or PRRSV (10^5–6 TCID₅₀/ml), with or without SNAP, respectively. At PTH 24 and 48, the cells were fixed in 95% ethanol and the TCID₅₀/ml was titrated by immunofluorescent staining using virus-specific monoclonal antibodies (SDOW-17 for PRRSV and 25H7/14E3 for PRCV). The TCID₅₀/ml was calculated according to the Reed and Muench method. All assays were performed independently three times. All data are expressed as the means ± SEM. Asterisks (*P < 0.05; **P < 0.01) indicate statistically significant differences between SNAP-treated, virus-infected and SNAP-untreated, virus-infected cells by unpaired Student's t-test.