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Role of Ganglioside GM1a in the infection by human rotaviruses.
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Role of Ganglioside GM1a in the infection by human rotaviruses.

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Category
Roles of glycans during microbial infection
Protocol Name

Role of Ganglioside GM1a in the infection by human rotaviruses.

Authors
Guo, Chao-Tan
Institute of Bioengineering, Zhejiang Academy of Medical Sciences

Suzuki, Yasuo *
Health Science Hills, College of Life and Health Sciences, Chubu University
*To whom correspondence should be addressed.
KeyWords
Reagents

Rota viruses:(Kun and MO strains) and feline (FRV64 strain) rotaviruses were propagated in embryonic rhesus monkey kidney cells (MA104 cells) as described previously 3).

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

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

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

Instruments

Microscopy

CO2 incubator

Methods
1.

Inhibitory activity of exogenous ganglioside GM1a on the Inhibition of MA104 cells by rotaviruses

1) 

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

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2) 

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

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3) 

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

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4) 

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

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5) 

 Add the ganglioside GM1a/virus mixture to MA104 cells, adsorb for 1 h at 37°C, and then remove and replace with the maintenance medium (MEM supplemented with 2% FBS) and leave for 16 h at 37°C, then wash the cells with PBS.

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6) 

 Determine the neutralization activity of exogenous GM1a by indirect immunostaining procedure as described next procedure.

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7) 

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

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8) 

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

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9) 

 Incubate the cells with PBS containing HRP-conjugated protein A at 37°C for 30 min, and stained with a solution containing 50 mM 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 three times with PBS.

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10) 

 Count the stained cells under light microscopy.

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Notes

Rota viruses should be handled in the facility of Biological Safety Level 2. Therefore, handling of the viruses should be under the control of national law.

Figure & Legends

Figure & Legends

Fig. 1. Inhibitory activity of exogenous ganglioside GM1a on the infection of MA104 cells by rotaviruses.

The inhibitory activity of GM1a (closed) and GA1 (asialo GM1) (open), which are used as controls for the infections KUN (triangle), MO (inverted triangle), and FRV64 (circle) strains of MA104 cells, was determined using a neutralization assay as described above.

Reprinted from J. Biochem., 126, Chao-Tan Guo, et al., Ganglioside GM1a on the cell surface is involved in the infection by human rotavirus KUN and MO strains, 683–688, 1999 with permission from The Japanese Biochemical Society.

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