Jpn. J. Infect. Dis., 53, 62166, 2000

Original Article

Effect of Sulfated Colominic Acid on Enteric Virus (RotaviruS, Poliovirus
and CoxsackieviruS) Infections In Vitro
Kyoko Konishi, Yanhong Gut, Ikumochi Hatano and Hiroshi Ushijimal *Department of Viral Diseases and Vaccine Contr10l, National Institute oflnfeclious Diseases,
MusashimmvJama‑Shi Too,0 208‑0011 andJDepartment ofDevelopmental Medical Sciences, Institute of International Health,Graduate School of Medicine. The UniversiO, of Too/0,
Hongo, Bunk,0‑ku, Too,0 113‑0033, Japan(Received January 28, 2000. Accepted May 1 I, 2000)
SUMMARY: Sulfated colominic acid exhibited suppressive effects on SA 1 1 (simian rotaviruS)‑ and MO (humanrotaviruS)‑infections, but not on Wa (human rotaviruS)‑, Sabin 1 (poliovirus 1)‑, and Nancy (Coxsackie B3 virus)‑
infections, in vitro. The hfection ofSAl 1 was found to be inhibited by mixed treatment and early posttreatment

with sulfated colominic acid, but not by pretreatment, by plaque assay and multiple growth assay. The results
were confirmed by the infectivity titer, RNA polyacrylamide gel electrophoresis, and electron microscopic analysis.
The mechanism of the suppressive effect was suggested to be adsorption inhibition at an early stage of the infection.

Colominic acid is a homopolymer of 〟‑acetylneuraminic

1NTRODUC TION

acid containing α‑2,8 ketosidic linkages between也e sugar

Rotaviruses are recognized as the major etiologlC agents Of

moieties (4). Recently, it has been reported that sulfateddiarrhea ininfants and young children. In developlng COunbies,
about 800,000 children die annually ofrotaviruSinfeCtion ( 1 ,2).

colominic acid exhibits anti‑HIV activity(5), and suppresses

To reduce the morbidity and mortality associated withneuronal cell death (6). Colominic acid is one or the sugar

rotavirus infection, intensive efforts made. Rotavirus live

chains, and reacts as siahc acid. Such sugar chains are present

PrPSc (scrapie prion protein) and HIV‑I gp1201induced

vaccine is one of the preventive methods, though a morein blood proteins and mammalian breast milk. They are

effective vaccine with fewer side effects still needs to berecognized as virus receptors on the cell surface (6). In thisdeveloped (3). AIthoughvarious formulations of oral rehydra‑

study, we examined the suppressive effects of sulfatedtion salt solutions have proved useful, they are insufrlCient

colominic acid on enteric vimses including rotavims, polio‑

for childrenwith severe and persistent diarrhea, especiallyvims, and coxsackie B3 vims.

for those with rotavirus diarrhea who have complications suchas viral encephalitis. An effective anti‑rotavirus drug might be

MATERIALS AND METHODS

beneficial for treatment, especially in immunocompromised
patients and malnourished children who are not recommended

Chemicals: Sulfated colominic acid was obtained fromto receive live rotavims vaccine or who have persistent

Mamkin Shoyu Co. Ltd. (Kyoto). The sodium salt with andia汀hea caused by rotavirus.

average molecular mass of24,000 (〃≒50) was used (Fig. 1).

R=H or SO3Na
Average molecular mass:24,000 (∩≒50)

Fig. 1. Structure ofsulfated colominic acid.

*Corresponding author: Tel/Fax: +81‑3‑5841‑3629, E‑mail:ushij ima@m. u‑tokyo. ac.jp

62inoculalion(I)I retreatm ent
prei ncu bation(ⅠⅠ)∩ ixed treatment

i nocublio rl(ⅢⅠ)I osttreatm ent
p rei mt:u batio n(IV)mixed p一us posttreatmontFig. 2. Schema offour treatments. SCA, V, and MEM represent sulfated colominic acid, virus,and Eagle's minimumessential
medium, respectively. ⊂=コindicates treatmentwithout cells, E:冨::ヨindicates treatment on the cells.

continuousincubation a触rinoculadon of virus bosttreabIent) ;MO. To the cultured cells, a 0.2 ml/tube or 100 TCID5｡ Of
MO was adsorbed for 1 h. Then, FBS‑free MEM supple‑
mented with 0. 1 5% bicarbonate and 1 LLg/ml ofacetyl trypsin
was added. Cell cytopathic effect was observed for 10 days.
Multiple step growth assay: One hundred pfu/0.2 ml ofSAll and 100 TCID5｡ /0.2 ml ofMO were inoculated into
tube cultures ofMA104 cells and inoculated for 1 h. Afterwashing the cells with PBS three times, the MEM containlngor 4) for I h withvirus beforeinOculationandunder continuous

0. 1 5% bicarbonate and 1 LLg/ml of acetyl trypsin was added.

The compound was dissolved in Ca2'and Mg2㌧free phosphate‑buffered saline (PBS(‑)) Containing 1 0% dimethyl sulfoxide atstock concentrationP of 1 0 mg/mland stored aト20oC unti1use･

Sulfated colomlnic acid, dilutedwith culture medium at
the time of experiment, was added to the medium as follows:I) at 24 h before inoculation of viruS (pretreatment); 2)for 1
h with vims befわre inoculation (mixed treatment); 3) under

When the cytopathic effect was detected in the positive

incubation after inoculation (mixed plus posttreatment)･ The
Inoculation time of the sulfated colominic acid‑treated virus

control cells (inゎ which the vims had been inoculated but noor sulfated colominic acid‑untreated virus was 1 h. After thesulfated colominic acid had been added), cells and culture

inoculation, the cells were washed with PBS(‑) three times

medium were separately frozen and thawed three times, thenfor infectivityassay, cell cytopathic assay, and multiple step

subsequently centrifuged at 6,000 X g for 5 min. The supema‑

growth assay. The treatments were conducted at 37oC, andtant nuid and cells were separately titered by plaque assay

the schema are exhibited in Figure 2.

cclls and viruses: MA104 cells derived from rhesus

for SAl 1 and cell cytopathic assay for MO (7).
RNA‑PAGE (Polyacrylamide gel electrophoresis):monkey kidney were culturedand passaged with Eagle'Smini‑

Rotavirus RNAsWere extracted from the supematant and themum essential medium (MEM) Containing 0. 1 5% bicarbonate
and lO% fetal bovine serum (FBS). Cultured‑rotaviruses

cells were separated by uslng Phenol and ch)oroform. Then(simian rotavirus SAl 1 , and human rotaviruses Wa and MO),segments in the gel were stained with silver (8).

each fraction was electrophoresed by Laemmli gel. The RNA‑poliovims se!otype 1 (Sabin 1), and ‑Coxsackie B3 virus

Titration ofviral proteins by enzyme immunoassay: The

culture supernatants and cells were examined for viral(Nancy) were lnOCulated to the cells (7).

Effect of sulfated colom!nic acid on cell yiability: MEM

proteins by uslng COmmerCially available immunoassay kits

contalnlng Sulfated colominlC acid was added to MA 1 04 cells
at different concentradons (0, 100, 200, 400, 800, or 1500 LL g/(Rotaclone, from BioSience, Cambridge, Mass., USA).

ml). The cells were cultured for 1 week. A洗er detaching cells

centrifuged at 6,000 X g for 20 min (partially purifiedviruS).

ElectronmicroscopIC analysis: The culturedviruses werewith trypsin, the numbers or viable cells were counted by

The supematant was ultracentrifuged at 100,000 X g for 1 h

nigrosin dye exclusion method (7). The experiment was

through 1 ml of35% sucrose in PBS. The pellet was resus‑

conducted in duplicate.

pended in PBS and recentrifuged at 100,000X g for 16 h in
CsCl. The virus bands were collected and dialyzed in PBS

Infectivity assay (direct plaque assay): One hundredplaque‑forming units (pfu) of rotaviruses (SAl 1 , Wa), polioI(purified virus)･ The purified virus particles †ere mixed with

400 LLg/ml or 0 LLg/ml ofsulfated colominlC acid at 35oC

vims 1, and coxsackie B3 Virus were adsorbed to cultured
cells at 0.2 ml/well on six‑well microplates f♭r 1 h. A触r

for 0, 10, and 60 min, respectively. The treated viruses were

washing with PBS, the cells were overlaid with agar contain‑

negatively stained with 2% uranyl acetate (pH 4.4) and viewed
with an electron microscope (model JEOL 100 CX) (8).

Ing FBS‑free MEM supplemented with 0. 1 5% bicarbonate,
0.6% agarose, and 0.002% neutral red. One LLg/ml of acetyl
trypsln‑treated rotaviruses were used in､the experiments.

RESUIXS

Sulfated colomimic acid was added to the medium at concen‑

Cytotoxicity: It was found that 1 ,500 LLg/ml of sulfatedtrations of 0, 50, 100, 200, and 400 LLg/ml, as indicated.

Cell cytopathic assay: For MO, the plaque assay was notcolominic acid reduced the number ofviable cells by 50%
and 800 LLg/ml reduced the number by 30%. No reduction of

successful because the plaque was not visible. Therefore, theviable cells was found at concentrations below 400 jig/ml

cell cytopathic assay was used for the infectivlty assay Of(Tab)e 1).

Experiments were perf♭med in duplicate (7).

63

Table 1. Cytotoxic effect of sulfated colominic acid

Table 3･ Effect of sulfated colominic acid on rotavirus in a multiple

on MA104 cells for 7 days

step growth assay

Concentration viable cell日‖ =O LLg/ml

0 0 9 8 7 5 0 0 7 /hV 2 1

virus

method

O

50

100

200

400

LLg/ml

SAll

%

loo

Pretreatment

200

mixed treatment

400

POSttreatment

800

7 7 7 7 1｡2 2 1′‑U 丘U 4 5

7 5 ′0 5

7 ∠U つJ つJ 2 tJ 7 7

7 ∠U tJ 3

422 777

4 2 L2 2 4

4 L L2 4 40 tJ 7 7

7 ∠U つJ つJ

3375

mixed plus posttreatment

1500

MO
pretreatment

444 747

4 L L2 2 2

mixed treatment

Table 2･ Effect ofsulfated colominic acid on enteric virusesinplaque assay
vlmS

methodO

SA 1 1 pretreatment

50

100
OO 5 42U 7 tJ

1 1 1 0 0 00 0 0200

po sttreatment

400〟g/ml

AV 4 29 4 ∠U

Data show thevirus yields (log ofpfu/0.2 ml in SAl 1 and log ofTCID5./
0.2ml in MO). One hundred pfuofSAl I/0.2 ml and loo TCID5. OfMO/

7 2 25 7 ′‑U

7 つJ 20 1 9

0.2 ml per tube were inoculated into MA1 04 cclls/Treatment with sulfatedmixed treatment

colominic acid and assays wereperformed as described in Materials andMethods.

posttreatment

Wa pretreatment

1 1 1 0 0 00 0 0

1

1

0 00 0 0 5 0

1 1 1 0 2 00 5 0

0 0ノ 00 ′0 0

1 0 0ノ 9 0 2 4ll 0 2 0ノ

1 1 1 O 1 1 0 tJ OO

00 つJ 2

mixed treatment

at posttreatment, virus growth and extracellular virus release

POSttreatment
Sabin I pretreatment

1 1 1 0 0 00 0 0

1

1

1

1

0 9 00 8 0

1

1

were delayed. Mixed treatment at 400 LLg/ml seemed to be

O 9 0 0 ノーU 0

effective until 2 days after inoculation, and in effective at 3

mixed treatmentdays (Table 4).

PO Sttreatment

Nancy pretreatment

ElectronmicroscopIC analysis: The aggregation of SA I I
111

Oノ 9 00 0 00 0 0

0 ′▲U 00ノ 0ノ 0ノ579

0ノ 0ノ 90ノ 9 0 つJ 5 0559

by sulfated colominic acid was examined under an electron

mixed treatment
posttreatment

micros.C?pe･ Aggregation was not seen at 0 LL g/ml of sulfated‑‑colomlnlC acid at 35oC at 0, 10,and 60 min, or at 400LLg/ml

Data are glVen aS PerCentageS Ofthe number ofplaques in the assay

at 35oC at 0 min. However, 2 to 3 particle aggregations were

containing the compound compared to assays without sulfated colominic

noted at 35oC, 10 min, and 5 to 10 particle aggregations wereacid (set to 1 OO%). One hundred p丘JO.2 ml of virus were inoculated into

seen at 35oC, I hr,underthe condition of400 LL g/ml (Fig. 3).

MA1 04 cells in each well ofa six‑well microplate. Treahentwith sulfatedcolominic acid was performed as described in Materialsand Methods.

DISCUSSION
Effects on viral infection by plaque assay and multiple

Outer capsid protein VP4 is the main rotavirus cell attach‑

growth assay: The antiviral effect of sulfated colominic acidment protein, but the cellular receptor remains unknown.

was e.xamined by inoculation of loo pfu of SAl 1 (simian

Sialic acid on the cell surface has been shown to be required

rotavlruS), Wa (human rotavims), MO (human rotavirus),

for efrlCient binding and infectivity of animal rotaviruses

Sabin 1 (poliovirus 1), and Nancy (Coxsackie B3 virus),including SAl 1 , but not of human rotavimses including Wa

respectively. Pretreatment at 50 to 400 〝g/ml reduced the(9‑1 1). MO has not been examined in previous reports. For

grow仇ofSAll by 12 to 31%; mixed treatment at 50 to 400

demonstration of sialic acid‑dependent or ‑independent〝g/ml reduced it by 43 to 73%; and pos肘eatment at 50 torotaviruS, neuraminidase‑treated MA 1 04 cells were used (91400 LL g/ml reduced it by 57 to 74%. These results show that

1 1)･ In our experiments, sulfated colominic acid, like sialic

both the mixed and the posttreatment reduced SA I I rotavirusacid, inhibited anlmal rotaviruS (SAl 1) and humaT rotaviruS

infection remarkably. The pretreatment, mixed treatment, andposttreatment at different concentrations of sulfated colominic(MO) infections but did not inhibit human rotavlruS infec‑
tion (Wa). This inhibition was shown in response to mixed

acid did not reduce viral titers of Sabin 1 , Nancy or Wa (Table

treatment and posttreatment. These findings indicate that2).
In multiple step growth assay, the pretreatment, mixed treat‑sulfated colominic acid is effective on virus adsorption at thetime of and/Or after the inoculation of SAl I and MO. Inhibi‑ment, posttreatment, and mixed plus posttreatment reduced

tion after inoculation was successful at an ear)y stage ofplaque numbers ofSAll by 0‑, 10‑, 10315‑ and lO316‑fold,infection; the infection was not inhibited when sulfatedrespectively. The mixed plus posttreatment did not show

colominic acid was added 5 h after the virus inoculation (datamultiplier effects on SAl 1. Themixed treatment and post‑

not shown). Our results present two problems yet to betreatment reduced plaque numbers of MO by lO3･2‑ and 1 O315‑

resolved. 1) In addition to sialic acid, there may be anotherfold, respectively (Table 3).

receptor for rotaviruS, such as gangliosides ( 1 2). The presence

The infectivity titer of SAll on the infected cells andthe culture supernatants: In order to examine the antiviralof a coreceptor may be necessary for some types ofrotavirus

mechanism of su脆ted colominic acid,血e vims growth was

may suggest that, in addition to the inhibition of virus

infection. 2) Inhibition ofrotavirus infection by posttreatment

examined separately in cells and supematants. By posttreat‑

adsorption by sulfated colominic acid, other mechanisms may‑

ment, the appearance of cell cytopathic effect and extracellu‑

Contribute. Given this possibilit汎We examined intracellular

lar release of the virus were delayed. The mixed treatment atand extracellular double‑stranded RNA and virus protein. No

400 LLg/ml had no effect at 2 days aRer infection,virus growth

Enzyme lmmtlnOaSSay and RNA‑PAGE: According tomeaningful differences were observed. Sulfated colominicacid by posttreatment might block the path of virus infection
frominfected cells to healthy cells. The results of electron

the increase of the concentration of sulfated colominic acidmicroscopic examination revealed that SA 1 1 reacted directly

was the sam.e as that of the positive control (Table 4)I64Table 4. Effect of sulfated colominic acid on multiplication of SAl 1 by posttreatment and mixed treatment
posttreatment mix ed treatment
O

concentration

50 jig/m1

nuid cell

fluid

3.4

2.2

3.6

0.03

0.04

cell

200 LLg/m1
fluid

eel

400 LLg/ml

l

flui

2.2

2.5

0 0 70 1

0.03

0.04

d

eel

I

lday CPE
log pfu/m1

0D

3.7

0.03

0.04

つJ

RNA ‑PAGE
2days CPE

+ .6 .7 +log pfu/ml

5 0

1｡2.+
00

OD

･qU .+

つJ 0

5 0+ <...

tJ 0

4 0

50 l

∠U 0
50 l

5 0

OO ‑
/▲U

00

5

0ノ

4

1

RNA ‑FAG E}6･3日5{

3days CPE

log pfu/ml

6･31 ･67}′▲U 1

つJ 0 rT IT: +

5 0
つJ

OD

}6･5̲ ･2 +･0 .1 +

+ .1 .6 +5 0 .5 月 +

5 0
∠U

9

つJ

00 3 +00

RNA ‑PA GE
4days CPE++

log pfu/m1

0D

1.62

RNA‑PAGE

6.4

6.3

1.50

〜

〜

++

+

6.0

6.3

4.8

5.8

1.34

1.48

0.30

0.75

++

++

++

NNNN DDDD+

One hundred pfuof SAl I was inoculated into MA104 cells. Culture fluids and cells wereanalysed separatelyby cell cytopathic effect (CPE), plaque‑forming unit bfu), enzyme immunoassay (OD values), and RNA
polyacrylamide gel electrophoresis (RNA‑PAGE). ‑, +,and ++ correspond to negative, visible,and strongly
visible, respectively.

Fig. 3. Electron micrograph of purified SAl I rotavirus after the four respective treatments. The virus was kept without

sulfated colominic acid for 1 h at 35oC (A). The virus was treated with 400 LLg/ml of sulfated colominic acid at 35oC, 0 min(B); 10min(C) and 1 h (D).

with sulfated colominic acid. These phenomena indicatethat

which may react to the sialic receptor of host cells, but is not

sulfated colominic acid behaves like sialic acid with regard

effective on infection of sialic acid‑independent rotaviruses,

to virus infection.

polioviruses, and coxsackieviruses, which do not have a ligand

nle reCq)tor Ofpoliovirus isknownto belong toanimmun0‑

to the sialic receptor of host cells. Further studies are needed

globulin super family (13)･ The recepto.rs of coxsackie B3
virus are the coxsackievirus and adenovlruS reCePtOr (CAR,

to confirm the relationship between virus and cell receptor.

a member of immunoglobulin super family) (14) and the
decay‑accelerating receptor (DAF) ( 1 5)･ Sulfated colominicacid might not be effective on poliovirus and coxsackievlruS
because of the difference in cell receptors.

ACKNOWLEDGMENT
This study was partly supported by a grand‑in‑aid from

the Ministry of Education, Science, Sports and Culture of

Our results show that sulfated colominic acid may beeffective on infection of sialic acid‑dependent rotaviruses,

Japan.

65

Tbkyo. ∫. Med.Viro1., 27, 299‑303.

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