Role of ganglioside GM1a in infection by human rotaviruses

Rotaviruses: Human (KUN and MO strains) and feline (FRV64 strain) rotaviruses were propagated in embryonic rhesus monkey kidney cells (MA104 cells) as described previously (3) (Note 1).

Gangliosides: Native gangliosides can be isolated from various tissues. Ganglio-series gangliosides, such as GM1a, GM1b, GD1a, GD1b, GT1a, GT1b, and GQ1b are mainly found in and taken from brains of mammals, including bovine, pig, and horse using anion-exchange and iatrobeads column chromatography (3). GA1 was prepared from GM1a.

Antibodies: Guinea pig anti-FRV-64 antiserum was prepared as follows: Four weeks-old pathogen-free guinea pigs were immunized with purified feline rotavirus FRV64 strain by intramuscular injection. One month later, the guinea pigs were boosted with a similar intraperitoneal injection. The sera of immunized guinea pigs (anti-FRV64 antisera) were harvested and purified after one week. The antisera crossreacted with human rotaviruses KUN and MO strains (3).

Horse radish peroxidase (HRP)-conjugated protein A was purchased from Organon Teknika N.V. Cappel Products.

Microscopy

CO₂ incubator

Preparation of cells, rotaviruses, and gangliosides

a.

MA104 cells were grown in MEM supplemented with 10% fetal bovine serum (FBS) at 37°C in a humidified 95% air, 5% CO₂ incubator.

b.

The viruses were titrated to determine a dilution giving ~200 staining focus-forming units per well of 96-well microtiter tray.

c.

Exogenous ganglioside GM1a (starting concentration, 50 μM) was serially diluted with PBS (20 mM of phosphate-buffered saline, pH 7.0).

Pretreatment, viral adsorption, and treatment

a.

Mix ganglioside GM1a with an equal volume of trypsin-activated viruses (preincubated with 10 μg/mL of trypsin for 30 min at 37°C) and maintain for 1 h at 37°C.

b.

Add the ganglioside GM1a/virus mixture to MA104 cells and adsorb for 1 h at 37°C.

c.

Replace the ganglioside GM1a/virus mixture with the maintenance medium (MEM supplemented with 2% FBS) and leave for 16 h at 37°C. Then, wash the cells with PBS.

Determination of GM1a inhibitory activity by an indirect immunostaining procedure (for example data, see Figure 1)

a.

Fix the infected MA104 cells 80% acetone for 10 min and wash thrice with PBS.

b.

Incubate the fixed cells with PBS containing anti-FRV64 antiserum at 37°C for 30 min. Then, wash the cells with PBS.

c.

Incubate the cells with PBS containing HRP-conjugated protein A at 37°C for 30 min and stained with a solution containing 50 mM of Tris buffer (pH 7.5), 0.5 mg/mL of 3.3’-diaminobenzidine tetrahydrochloride, and 0.01% H₂O₂ aqueous for 20 min at 4°C and wash thrice with PBS.

d.

Count the stained cells under light microscopy.

Rotaviruses should be handled in the facility of Biological Safety Level 2 under the control of national law (6).