Infection of the tracheal epithelium by infectious bronchitis virus is sialic acid dependent - PubMed (original) (raw)

Infection of the tracheal epithelium by infectious bronchitis virus is sialic acid dependent

Christine Winter et al. Microbes Infect. 2008 Apr.

Abstract

Avian Infectious bronchitis virus (IBV) is a coronavirus that infects chickens via the respiratory epithelium as primary target cells. The binding of coronaviruses to the cell surface is mediated by the viral surface protein S. Recently we demonstrated that alpha2,3-linked sialic acid serves as a receptor determinant for IBV on Vero cells and primary chicken embryo kidney cells. Here we analyze the importance of the sialic acid binding activity for the infection of tracheal organ cultures (TOCs) by different IBV strains. Our results show that alpha2,3-linked sialic acid also serves as a receptor determinant on chicken TOCs. Infection of TOCs by IBV results in ciliostasis. Desialylation induced by neuraminidase treatment of tracheal organ cultures prior to infection by IBV delayed the ciliostatic effect or resulted in partial loss of ciliary activity. This effect was observed with both respiratory and nephropathogenic strains. Inhibition of ciliostasis was also observed when TOCs were pretreated with an alpha2,3-specific neuraminidase. Analysis of the tracheal epithelium for reactivity with lectins revealed that the susceptible cells in the epithelium abundantly express alpha2,3-linked sialic acid. These results indicate that alpha2,3-linked sialic acid plays an important role for infection of the respiratory epithelium by IBV.

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Figures

Fig. 1

Fig. 1

Effect of neuraminidase treatment on the infection of TOC by IBV. TOC were incubated in the absence (−NA) or presence (+NA) of neuraminidase. In two samples the enzyme activity was prevented by addition of the inhibitor DANA. Following neuraminidase treatment, the cultures were infected by IBV-Beaudette. The effect of the infection was estimated at different days post infection (d.p.i.), by determining the loss of the ciliary activity.

Fig. 2

Fig. 2

Effect of α2,3-specific neuraminidase on the infection of TOC by IBV. Tracheal rings were incubated with neuraminidase from Streptococcus pneumoniae prior to infection IBV-Beaudette. The effect of the virus infection was estimated by determining the loss of ciliary activity.

Fig. 3

Fig. 3

Infection of tracheal organ cultures by different IBV strains. TOC were incubated in the absence (−NA) or presence (+NA) of neuraminidase prior to mock-infection (control) or infection by IBV strains Beaudette (Bd), B1648 or M41 (panels A–C) or Avian metapneumovirus (panel D). The effect of the virus infection was estimated by determining the loss of ciliary activity.

Fig. 4

Fig. 4

Effect of pretreatment with neuraminidase on the virus release from IBV-infected TOC. TOC were incubated in the absence (−NA) or presence (+NA) of neuraminidase from Clostridium perfringens and then infected by either of two IBV strains, Beaudette or M41. At 24 h post infection, infectious virus in the supernatants was titrated by plaque assay on primary chicken kidney cells.

Fig. 5

Fig. 5

Immunofluorescence analysis of IBV-infected TOC. Cryosections were prepared from infected TOC and stained for virus antigen (green fluorescence in the top row and red fluorescence in rows 2–4). In addition, the samples were stained for the presence of ciliated cells (top row, anti-β-tubulin), of mucin-producing cells (second row, anti-MUC-5AC), cells expressing α2,3-linked sialic acids (third row, MAA), and cells expressing α2,6-linked sialic acids (bottom row, SNA).

Fig. 6

Fig. 6

Effect of neuraminidase treatment on the staining of the tracheal epithelium by the lectin MAA. TOCs were incubated in the presence (+NA) or absence (−NA) of neuraminidase. After incubation, cryosections were prepared and subjected to MAA staining. Arrows point to the apical membrane of the tracheal epithelium. It should be noted that the enzyme had access only to the apical side of the epithelium.

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