Antiviral effects of a probiotic Enterococcus faecium strain against transmissible gastroenteritis coronavirus

Weidong Chai, Michael Burwinkel, Zhenya Wang, Christiane Palissa, Bettina Esch, Sven Twardziok, Juliane Rieger, Paul Wrede, Michael F G Schmidt, Weidong Chai, Michael Burwinkel, Zhenya Wang, Christiane Palissa, Bettina Esch, Sven Twardziok, Juliane Rieger, Paul Wrede, Michael F G Schmidt

Abstract

The enteropathogenic coronavirus transmissible gastroenteritis virus (TGEV) causes severe disease in young piglets. We have studied the protective effects of the probiotic Enterococcus faecium NCIMB 10415 (E. faecium), which is approved as a feed additive in the European Union, against TGEV infection. E. faecium was added to swine testicle (ST) cells before, concomitantly with, or after TGEV infection. Viability assays revealed that E. faecium led to a dose-dependent rescue of viability of TGEV-infected cells reaching nearly to complete protection. Virus yields of the E. faecium-treated cultures were reduced by up to three log10 units. Western blot analysis of purified TGEV revealed that the levels of all viral structural proteins were reduced after E. faecium treatment. Using transmission electron microscopy, we observed attachment of TGEV particles to the surface of E. faecium which might be a means to trap virus and to prevent infection. Increased production of nitric oxide in the cells treated with E. faecium and elevated expression of interleukin 6 and 8 pointed to stimulated cellular defense as a mechanism to fight TGEV infection.

Figures

Fig. 1
Fig. 1
Effect of E. faecium on the viability of ST cells. E. faecium was added to confluent cells in a 96-well plate, which were then incubated at 37 °C for 1.5 h. Cell viability was tested by MTT assay after 72 h. The cell survival rate was determined by comparing the optical density values from E. faecium–treated cells to those from non-treated control cells set to 100 %. The cell survival rates at different concentrations of probiotic bacteria are given, and 50 % above the cell survival rate (above broken line) is regarded as a non-toxic concentration of E. faecium. Results represent means ± standard deviations from three independent experiments
Fig. 2
Fig. 2
Rescue of TGEV-infected ST cells by treatment with E. faecium. Different concentrations of E. faecium were added to ST cells in different setups as described (a). Heat-inactivated E. faecium (b) and diluted supernatants of cultured E. faecium (c) were also included. After 72 h, an MTT assay was carried out. Results are plotted as percent viability, with uninfected cells without E. faecium taken as 100 %. Results are given as mean ± standard deviation from at least three independent experiments
Fig. 3
Fig. 3
Attachment of TGEV particles to E. faecium. The pellet of virus and bacteria mixture from cell-free preincubation assay was negatively stained and examined by TEM
Fig. 4
Fig. 4
Less virus produced in E. faecium–treated ST cells infected with TGEV. Cells were exposed to different concentrations of E. faecium as described. Cell culture supernatants were collected, and the yield of virus was determined by TCID50. The means ± standard deviations from three independent experiments are shown. Significance levels for the difference between E. faecium treatment and virus control from untreated cells are given above the bar: **p < 0.001, *p < 0.05
Fig. 5
Fig. 5
Disrupted TGEV protein expression in E. faecium–treated ST cells infected with TGEV. Lane 1, uninfected, untreated cell control; lane 2, TGEV control; lane 3, virus from cells treated with 1.00E+05 CFU/ml E. faecium; lane 4, virus from cells treated with 1.00E+06 CFU/ml E. faecium; lane 5, virus from cells treated with 1.00E+07 CFU/ml E. faecium. Molecular weight marker proteins were run in parallel. Positions of viral spike protein (S), viral nucleocapsid protein (N), and viral membrane protein (M) are indicated on the right. Three independent experiments yielded almost identical results
Fig. 6
Fig. 6
Stimulation of cytokine expression by E. faecium in TGEV-infected ST cells. The expression of selected cytokines was measured by quantitative RT-PCR. The expression of IL-6 and IL-8 was significantly increased compared to the cells infected with TGEV only (black bars). Significance levels for the difference between E. faecium treatment and virus control are given above the bar: *p < 0.05, **p < 0.01. The data presented correspond to the mean ± SD of at least three independent experiments

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