The sialic acid binding activity of the S protein facilitates infection by porcine transmissible gastroenteritis coronavirus

Abstract

Background: Transmissible gastroenteritis virus (TGEV) has a sialic acid binding activity that is believed to be important for enteropathogenicity, but that has so far appeared to be dispensable for infection of cultured cells. The aims of this study were to determine the effect of sialic acid binding for the infection of cultured cells under unfavorable conditions, and comparison of TGEV strains and mutants, as well as the avian coronavirus IBV concerning their dependence on the sialic acid binding activity.

Methods: The infectivity of different viruses was analyzed by a plaque assay after adsorption times of 5, 20, and 60 min. Prior to infection, cultured cells were either treated with neuraminidase to deplete sialic acids from the cell surface, or mock-treated. In a second approach, pre-treatment of the virus with porcine intestinal mucin was performed, followed by the plaque assay after a 5 min adsorption time. A student's t-test was used to verify the significance of the results.

Results: Desialylation of cells only had a minor effect on the infection by TGEV strain Purdue 46 when an adsorption period of 60 min was allowed for initiation of infection. However, when the adsorption time was reduced to 5 min the infectivity on desialylated cells decreased by more than 60%. A TGEV PUR46 mutant (HAD3) deficient in sialic acid binding showed a 77% lower titer than the parental virus after a 5 min adsorption time. After an adsorption time of 60 min the titer of HAD3 was 58% lower than that of TGEV PUR46. Another TGEV strain, TGEV Miller, and IBV Beaudette showed a reduction in infectivity after neuraminidase treatment of the cultured cells irrespective of the virion adsorption time.

Conclusions: Our results suggest that the sialic acid binding activity facilitates the infection by TGEV under unfavorable environmental conditions. The dependence on the sialic acid binding activity for an efficient infection differs in the analyzed TGEV strains.

Figure 1

Sialic acid dependent infection by TGEV PUR46. The infectivity of parental virus (left columns) and the HAD3 mutant virus was determined for adsorption times of 5, 20 or 60 min, respectively. The m8 and m10 mutant viruses were analyzed for adsorption times of 5 and 60 minutes. Prior to infection, ST cells were incubated for 60 min with either PBS (grey columns) or PBS containing 50 mU of neuraminidase (white columns). All experiments were performed 4 times with standard deviations shown at the corresponding columns. Significant differences are marked with an asterisk (*, p-value < 0.05).

Figure 2

Sialic acid dependent infection by IBV Beaudette and TGEV Miller at short and long adsorption times. The infectivity of IBV Beaudette and TGEV Miller was determined at adsorption times of 5 or 60 min, respectively. Prior to infection, Vero cells (for IBV) and ST cells (for TGEV) were incubated for 60 min with either PBS (grey columns) or PBS containing 50 mU of neuraminidase (white columns). The experiments were performed 3 times. Standard deviations are indicated. Significant differences are marked with an asterisk (*, p-value < 0.05).

Figure 3

Comparison of early and late infectivity between TGEV PUR46, HAD3 and TGEV Miller. The infectious titers of TGEV PUR46, HAD3 and TGEV Miller were calculated at adsorption times of 5, 20 and 60 min, respectively. Infectivity was expressed in plaque forming units per ml (PFU/ml). Prior to infection, ST cells were incubated with either PBS (1, 4, 5) or PBS containing 50 mU of neuraminidase (2, 3, 6).

Figure 4

Mucin-dependent infection of TGEV PUR46. Prior to infection, TGEV PUR46 was treated with the indicated mucin concentrations for 30 min at room temperature. After a 5 min adsorption time of the virus-mucin-mixture a plaque assay was performed. Plaque reduction by mucin treatment was calculated out of 2 independent experiments with quadruplicates. Standard deviations are indicated. Significant differences are marked with an asterisk (*, p-value < 0.05).