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

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.

Inhibitory influence of Enterococcus faecium on the propagation of swine influenza A virus in vitro.

The control of infectious diseases such as swine influenza viruses (SwIV) plays an important role in food production both from the animal health and from the public health point of view. Probiotic microorganisms and other health improving food supplements have been given increasing attention in recent years, but, no information on the effects of probiotics on swine influenza virus is available. Here we address this question by assessing the inhibitory potential of the probiotic Enterococcus faecium NCIMB 10415 (E. faecium) on the replication of two porcine strains of influenza virus (H1N1 and H3N2 strain) in a continuous porcine macrophage cell line (3D4/21) and in MDBK cells. Cell cultures were treated with E. faecium at the non-toxic concentration of 1×10(6) CFU/ml in growth medium for 60 to 90 min before, during and after SwIV infection. After further incubation of cultures in probiotic-free growth medium, cell viability and virus propagation were determined at 48 h or 96 h post infection. The results obtained reveal an almost complete recovery of viability of SwIV infected cells and an inhibition of virus multiplication by up to four log units in the E. faecium treated cells. In both 3D4/21- and MDBK-cells a 60 min treatment with E. faecium stimulated nitric oxide (NO) release which is in line with published evidence for an antiviral function of NO. Furthermore, E. faecium caused a modified cellular expression of selected mediators of defence in 3D4-cells: while the expression of TNF-α, TLR-3 and IL-6 were decreased in the SwIV-infected and probiotic treated cells, IL-10 was found to be increased. Since we obtained experimental evidence for the direct adsorptive trapping of SwIV through E. faecium, this probiotic microorganism inhibits influenza viruses by at least two mechanisms, direct physical interaction and strengthening of innate defence at the cellular level.

Antiviral activity of zinc salts against transmissible gastroenteritis virus in vitro.

Zinc has been shown to mediate antiviral effects against certain viruses. However, the underlying mechanisms are still largely unknown. We investigated the effects of the two zinc salts, zinc chloride (ZnCl(2)) and zinc sulfate (ZnSO(4)), on infection of swine testicle (ST) cells with transmissible gastroenteritis virus (TGEV) and compared it to the effects of a control salt, magnesium sulfate (MgSO(4)). Virus yield reduction experiments showed that ZnCl(2) and ZnSO(4) did not exhibit direct virucidal effects and did not affect adsorption of TGEV to ST cells. However, ZnCl(2) and ZnSO(4) markedly reduced viral titers as well as TGEV RNA and viral protein synthesis when applied during virus penetration and at different time points after viral cell entry. The results of the study suggest that zinc salts do not interfere with TGEV-cell binding but that they mediate antiviral effects through inhibition of viral penetration or egress or the intracellular phase of the viral life-cycle.

Molecular and phenotypic modulations of primary and immortalized canine chondrocytes in different culture systems.

This study was conducted to determine physiological and functional features of primary and immortalized canine chondrocytes. Chondrocytes were immortalized by introducing the catalytic component of human telomerase namely human telomerase reverse transcriptase (hTERT). Primary chondrocytes lost their characteristic phenotype and growth properties whereas the immortalized cells remained polygonal with rapid growth rate. The expression of chondrocyte-specific markers decreased many-fold whereas that of chondrocyte-non-specific gene increased in primary chondrocytes. The immortalized cells did not express chondrocyte-specific genes in monolayers. Both primary and immortalized cells were encapsulated in alginate microspheres to construct three-dimensional (3D) culture system. As the primary chondrocytes, also the telomerase-transfected cells adopted a chondrocyte-specific gene expression pattern in alginate culture. Thus, the expression of telomerase represents possibility to expand chondrocytes without limitation while maintaining the chondrocyte-specific phenotype in 3D cultures. Use of such cells provides a standardized tool for testing different tissue engineering applications in canine model.