Microbiol. Immunol. Vol. 22 (2), 81-88, 1978
Common
Antigen between Coxsackievirus A 16 and Enterovirus 71
Akio HAGIWARA, Isamu
TAGAYA, and
Tetsuo
YONEYAMA
Departmentof Enteroviruses,National Institute of Health, Tokyo (Received for publication, September 20, 1977)
Abstract Cross immunofluorescence revealed that coxsackievirus A 16 (CA 16) shared a common antigen with enterovirus 71 (E 71). The cross reactivity of these two serotypes was also examined by complement fixation test with purified virus preparations fractionated by sucrose density gradient centrifugation and two peaks of antigenicity were detected, one being type-specific and the other cross-reacting. The common antigen was heat-stable and attributable to empty capsids. Immunodiffusion also revealed the common antigen. Infants without antibody to E 71 developed complement fixing and precipitin antibody to E 71 after recovery from hand, foot and mouth disease caused by CA 16.
A widespread epidemic of hand, foot and mouth disease (HFMD) took place in 1973 in Japan and the causative agent was identified as E 71 (8). While we had been attempting to identify viruses isolated from patients in this epidemic, we noticed that virus isolates had a common antigen with CA 16 detectable by immunofluorescence. There was no neutralizability between these isolates and CA 16, and the isolates were finally identified as E 71. Further studies were carried out to clarify the antigenic relationship between these two serotypes and the results indicated that they share a common antigen attributable to empty capsids. MATERIALS
AND
METHODS
Cells. A continuous line of cynomolgus monkey cells established in our laboratory which was designated as CMK was used in most experiments. CMK cells were maintained in Eagle's minimal essential medium (Nissui Seiyaku Co.) with 10% bovine serum, 0.15% sodium bicarbonate and antibiotics. Primary cultures of cynomolgus monkey kidney cells (MK) were also used for virus preparation, which were grown in Earle's balanced salt solution with 0.5% lactalbumin hydrolysate and 2% bovine serum and antibiotics. Infected cells were maintained with serum-free medium. Viruses. Nagoya strain, a representative of E 71 isolates in 1973, was used most extensively. The prototype BrCr strain of E 71 was also used. The passage history of these two strains appeared elsewhere (8). The prototype G-10 strain of CA 16, originally received from CDC, Atlanta, USA, was passed from a homogenate of infected suckling mouse torsos to MK cells and then passed through CMK cells 81
82
A. HAGIWARA
12
to
14
times.
No
antigenic
change
was
ET AL
observed
during
the
monkey
kidney
cell
passages. Monolayers (ca. 0.1 maximum. for
PFU/cell) The
5 min
centrated on
a
of
cells
were
infected
with
an
and the cultures were harvested harvest was frozen and thawed
and
then
with
polyethylene
cushion
rotor.
CMK
of
centrifuged
CsCl
at glycol
(density
Band-formed
3,000
1.43)
virus
was
rpm
and at
10
min.
The
for
by
and
albino
supernate
through
rpm
purified
diluted
virus
cytopathic effect reached a times, sonicated at 10 MHz
centrifuged
40,000
further
when five
for
then
appropriately
90
CsCl
min
in
was
a Spinco
centrifugation
con-
(density
in
a
1.31) SW
CsCl
50.1
density
gradient. Immune G-10 each
of
crude
virus
virus
given
suspension
second mals
were
with
the
J.
immune
7 days
G-10 This
71
was
labeled
Sucrose the
top
pH 7.2 Fractions
a
linear
(12).
were
of put
a moistened
and purified
of the
injection. E
71
mixture
USA.
also
used
as
described
procedure
appeared G-10
16
in
and
a of
Ten
Purified
gradient
(15
was
to
carried
concentrated ml
virus
40%
of
out by
w/v)
days
serum the
later
the
The
ani-
prepared
courtesy fluid
WHO
of
Dr.
prepared
in
reference
horse
experiments. (8).
previous CA
(0.5 in
melted
1%
by
box
at
a
16
publication or
to
1.0
Nagoya
Noble
room
agar
ml)
veronal
(7). strain
in in
rpm
of
3 mm diameter the reagents
layered
density buffered
were cut were added,
on
saline
for
in
a CsCl
veronal
was
buffered
microtechnique
centrifugation
(10 •~•@ 12 cm). Wells of neighboring wells. After plastic
30
of the
isothiocyanate.
This were
The
amounts
ascitic
elsewhere
strain
days
suspension.
by
some
in
30
monkey
mice
CA
performed
with
and
available
of
5 ml
injection
equal
virus
with of
preparation.
of
immune
immune
strain
were
the
made
An
G-10
of
An
was
virus
(Difco),
2 ml
immunized injections
a booster
in a Spinco SW 50.1 rotor at 40,000 dropwise from the bottom of the tube.
fixation.
on a glass plate 5 mm between
the
were
subcutaneous
adjuvant
centrifugation.
sucrose
Antigens
in
of
fluorescein
gradient
Immunodiffusion. placed tance
last
prepared
and centrifuged were collected
Complement tray
with
density
of
The globulin
4 ml
with
strain
was
Immunofluorescence.
E
the
rabbits 4
days of
complete
prototype
against
immune
with
California,
Neutralization.
Monkey
injections
strain
the
given
alternate
intravenously
after
BrCr
with
serum
two
given
Schmidt,
laboratory
on
Freund's
was
bled
were
intramuscularly
prototype
Nathalie
given
and
injection
Monkeys
preparation
were
was
monkeys
strains.
Rabbits shot
our
Cynomolgus
Nagoya
the
later. first
sera.
and
2hr
a
at
at 5 C.
disposable gradient. saline with the
was a displates
temperature.
RESULTS
CrossImmunofluorescence betweenCA 16 and E 71 CMK cells infected with Nagoya strain were examined with labeled globulin against G-10 strain. Specific antigens were detected in the cytoplasm of infected cells (Fig. la). The specificity of the reaction was confirmed by inhibition of the staining with type-specific non-labeled immune serum as well as negative staining of non-infected cells and cells infected with the other coxsackievirus A or B which can
COMMON
ANTIGEN
BETWEEN CA
16 AND
E 71
83
a
b
Fig. I a. lmmunofluorescent staining of Nagoya strain-infected cells with an immune monkey globulin prepared with G-10 strain of CA 16. Fig. lb. I mmunofluoreseent staining of G-10 strain-infected cells with an immune monkey globulin prepared with Nagoya strain of 71.
grow in CMK cells and with several echovirus serotypes. The staining titer of this globulin for specific bright fluorescence was 1:4, while it was I :8 in CMK cells infected with the homologous virus. When CMK cells infected with the prototype BrCr of E 71 were examined, the same staining titer of 1:4 was obtained. CMK cells infected with G-10 strain could also be stained with the labeled globulin against Nagoya strain (Fig. lb), but in this case the staining titer was 1:1 (undiluted), while the homologous staining titer was 1:8. Twenty isolates of E71 in 1973 sent from various laboratories were also positive when tested for staining with the labeled globulin against G-10 strain. CrossReactionbetweenCA 16 and E 71 by ComplementFixation Preliminary experiments by use of an immune mouse ascitic fluid prepared with
84
A. HAGIWARA
ET AL
G-10 strain of CA 16 suggested that Nagoya strain shared a common complementfixing (CF) antigen with CA 16. The CF reactivity of the fractions of G-10 or Nagoya strain obtained by centrifugation in a sucrose density gradient was examined with the immune rabbit sera prepared with G-10 or Nagoya strain. As shown in Fig. 2a, two peaks of CF reactivity were shown when fractions of G-10 strain were examined with the homologous antiserum, which corresponded to the visible C and D bands reported with coxsackie- and polioviruses (6, 13). On the contrary, only a
b
Fig.
2. G-10
Complement-fixing strain
obtained
centrifugation
against
rologous sera. 30
activity by the
(anti-Nagoya a.
Native
min. •œ,
CF
rabbit
immune
against
an
sucrose
virus.
b.
Virus
titer
and
heated
CF rabbit
of
gradient
rabbit
against
serum; •›,
anti-Nagoya
fractions
homologous strain)
antigen
of density
hete-
immune at
56
C for
a homologous antigen immune
titer serum.
COMMON
ANTIGEN
BETWEEN
CA
16 AND
85
E 71
one peak, corresponding to the C band, was observed when the same fractions were examined with the anti-Nagoya serum. When the virus preparation was heated at 56 C for 30 min before centrifugation, the D band and the corresponding CF peak detectable with the homologous immune serum disappeared, while the fractions corresponding to the C band still showed a high CF reactivity against both the homologous and heterologous immune sera (Fig. 2b). A similar pattern of CF reactivity was shown with fractions obtained by centrifugation of Nagoya strain against the homologous and heterologous immune sera (data not shown). It is thus shown that empty capsids of CA 16 and E 71 share a common antigen reactive in the complement fixation test. Table
1.
Antibody
responses
of infants
after
the
primary
infection
with
CA
16
The antibody response of young children after the primary infection with CA 16 corroborated the above observations. In 1975 there observed some outbreaks of HFMD in Japan and viruses isolated from patients were identified as CA 16. We could examine paired sera of two patients born after 1973. As shown in Table 1, these children showed rises of both neutralizing and CF antibodies against CA 16 after recovery. When these sera were examined with E 71, only CF antibody was shown in the convalescent sera. Similar findings were also reported by Tokuda (17). CommonAntigenbetweenCA 16 and E 71 as Shownby Immunodiffusion Monkey immune sera prepared with G-10, Nagoya and BrCr strains were used, whose homotypic neutralizing antibody titered 1:500, 1:1,100 and 1:4,500 respectively. Paired sera of one of the HFMD patients used in the preceding experiment were also included. The native antigen was purified and concentrated by centrifugation in a CsCl solution about 1,000-fold by infectivity. A heated antigen was prepared by heating the native antigen at 60 C for 20 min. As shown in Fig. 3a, the convalescent serum of a patient showed the type-specific band with the native G-10 antigen and also the group-specific precipitin band with the native antigen of Nagoya and BrCr strains. This band appeared to coalesce with the band formed
86
A. HAGIWARA
ET Al,
a
b
c
Fig. 3. Cross immunodiffusion between CA 16 and E 71. Contents of holes: 1 =acute-phase serum from a CA 16 patient, 2 =Nagoya immune monkey serum, 3 = convalescent-phase serum from a CA 16 patient, 4 =G-10 immune monkey serum, 5= BrCr immune monkey serum (given by Dr. N.J. Schmidt), 6=native Nagoya strain, 7 =heated Nagoya strain, 8= native G-10 strain, 9= heated G-10 strain, 10= native BrCr strain.
between this serum and the native G-10 strain, but the continuity of the groupspecific band was not necessarily clear due to the overlapping of the type-specific and group-specific bands between the serum and the homologous antigen. When the heated G-10 antigen was used, the precipitin band due to the common antigen between the two serotypes was clearly observed as shown in Fig. 3b. Another experiment shown in Fig. 3c indicated that the band formed between the heated Nagoya antigen and the convalescent serum of the patient coalesced with those formed between the same antigen and the homologous anti-E 71 monkey sera as well as the monkey immune serum against CA 16 (G-10). The type-specific band formed between the native Nagoya virus and the homologous immune monkey sera crossed with the former group-specific precipitin band. DISCUSSION It has been established that the main etiologic agent of HFMD is CA 16. Recently two big epidemics of HFMD took place in Japan: one in 1969-70 was caused by CA 16, but the other in 1973 was proved to be due to E 71 infection as reported previously (8). During the course of identifying virus isolates, which later proved to be E 71, it was shown that they had a common antigen with CA 16 detectable by immunofluorescence. Although these two serotypes had no cross neutralizability, it was considered necessary to clarify the antigenic relationship between the
COMMON
ANTIGEN
BETWEEN
CA
16 AND
E 71
87
two serotypes because they brought about the same clinical symptoms. The results presented here have indicated that these two serotypes share common antigenicity attributable to empty capsids, which can be detected by immunofluorescence, complement fixation and immunodiffusion. Group antigen was demonstrated among coxsackievirus group B serotypes and group A type 9 by immunodiffusion (15, 16). Balayan et al studied the precipitin test with echovirus types 1, 6, 8, 13 and 19 as well as CA 7 and 9, and only CA 7 and 9 showed two precipitin lines with the respective standard monkey immune serum, one of them corresponding to the common antigen and the second to the type-specific antigen (1). Echovirus types 1 and 8 are antigenically closely related by cross neutralization tests and share common precipitin antigen(s) besides respective type-specific antigen (14). Conant et al studied echovirus 4 and other enteroviruses by gel precipitation and found that a serologic variant of echovirus 4, Shropshire and the prototype strain Pesascek shared a common precipitin antigen as well as type-specific antigens, which, however, did not appear to be completely identical, but with some strain specificity (3). From the results with coxsackievirus group B type 6 and echovirus 14, they suggested the existence of a group antigen shared by human enteroviruses, but the mutual relationship of their "B" lines was not clarified . With three types of poliovirus the different antigenicity of empty capsid from complete virion has been shown by complement fixation and immunodiffusion, but both antigens were type specific with immune animal sera (10, 11). Some human sera with monotypic neutralizing antibody reacted with heterotypic "C" or "H" antigens by complement-fixation, but other sera did not (10). Hinuma and Hummeler (9) reported that immunofluorescence of poliovirus was type-specific, although the antigens responsible for immunofluorescence were proved to be "H". Among coxsackievirus B serotypes French et al (5) reported that there observed some heterotypic and nonspecific staining (diffuse 1-2 + staining) at low serum dilutions by indirect immunofluorescence, but the immune reagents could be diluted to a point where they gave no heterotypic reactivity, but still showed characteristic homotypic staining. By direct immunofluorescence we observed that CMK cells infected with E 71 could be stained specifically with a labeled immune globulin against CA 16. As shown in Fig. 1a, brightly staining aggregates of viral antigen in the cytoplasm of infected cells were observed, and the staining titer of this globulin for 3-4 + staining was 1 :8 for CA 16-infected cells and 1:4 for E 71-infected cells. On the contrary, a labeled immune globulin prepared with Nagoya strain was more type-specific, the homologous staining titer being 1 :8, while the heterotypic titer was 1:1. In this case, the heterotypic staining was also specific with bright aggregates of antigen in the cytoplasm (Fig. 1b). The results obtained in the present experiments seem to indicate that the relationship between CA 16 and E 71 may be closer than among three types of poliovirus or among coxsackievirus group B and group A type 9. Classification of enteroviruses is based on neutralization, but three types of poliovirus are classified as one subgroup, while echovirus 1 and 8, which are related even by neutralization, are still grouped as independent serotypes (4). Some cross reactivity among several cox-
88
A. HAGIWARA
ET AL
sackievirus A serotypes has not been fully studied (2). Due to various biological and serological properties it may be reasonable to speculate that three serotypes of poliovirus might have evolved from the same root. In this connection CA 16 and E 71 may form another subgroup among enteroviruses. It may be of interest to study other enterovirus serotypes with an attempt of subgrouping some of them on the basis of common antigen as well as by the grade of homology of nucleic acids. We acknowledgethe courtesyof Drs. M. Tokuda, Institutefor VirusResearch,KyotoUniversity,and C. Miwa,GifuPrefecturalInstituteof PublicHealth,for providingus the pairedseraof HFMDpatients. REFERENCES
1)
Balayan, M.S., Belyaeva, A.P., and Seibel, V.B. 1963. Use of the precipitin test in the diagnosis of infections caused by ECHO and Coxsackie viruses. Acta Virol. 7: 241-249. 2) Committee on enteroviruses. 1962. Classification of human enteroviruses. Virol. 16: 501-504. 3) Conant, R.M., Barron, A.L., and Milgrom, F. 1966. Gel precipitation with echovirus 4 and other enteroviruses. Proc Soc. Exp. Biol. Med. 148: 203-207. 4) Fenner, F. 1976. Classification and nomenclature of viruses; Picornaviridae. Intervirology 7: 39. 5) French, M.L.V., Schmidt, N.J., Emmons, R.W., and Lennette, E.H. 1972. Immunofluorescence staining of group B coxsackievirus. Appl. Microbiol. 23: 54-61. 6) Frommhagens, L. 1965. The separation and physicochemical properties of the C and D antigens of coxsackievirus. J. Immunol. 95: 818-822. 7) Hagiwara, A., and Kitahara, T. 1974. Enhancing effect of green monkey kidney cell extract on the transformation of mouse cells by SV40. Int. J. Cancer 14: 26-31. 8) Hagiwara, A., Tagaya, I., and Yoneyama, T. 1978. Epidemic of hand, foot and mouth disease associated with enterovirus 71 infection. Intervirology 9: 60-63. 9) Hinuma, Y., and Hummeler, K. 1961. Studies on the complement-fixing antigens of poliomyelitis. III. Intracellular development of antigen. J. Immunol. 87: 367-375. 10) Hummeler, K., and Hamparian, V.V. 1958. Studies on the complement-fixing antigens of poliomyelitis. I. Demonstration of type and group specific antigens in native and heated viral preparations. J. Immunol. 81: 499-505. 11) Le Bouvier, G.L. 1959. Poliovirus D and C antigens: Their differentiation and measurement by precipitation in agar., Brit. J. Exp. Pathol. 40: 452-462. 12) Lennette, E.H. 1969. Complement fixation test. p. 52-58. In Diagnostic Procedures for viral and Rickttsial Infections, 4th ed, American Public Health Association, Inc. 13) Mayer, M.M., Rapp, H.J., Roizman, B., Klein, S.W., Crowan, K.M., Kukens, D., Schwerdt, F.L., and Charney, J., 1955. The purification of poliomyelitis virus as studied by complement fixation. J. Immunol. 78: 435-455. 14) Middleton, G.K., Jr., Cramblett, H.G., Moffet, H.L., Black, J.P., and Shulenberger, H. 1964. Micro diffusion precipitin tests for enteroviruses and influenza B virus. J. Bacteriol. 87: 11711176. 15) Schmidt, N.J., and Lennette, E.H. 1962. Gel double diffusion studies with group B and group A, type 9 coxsackie viruses. I. The technique and reactions obtained with hyperimmune animal sera and human sera. J. Immunol. 89: 85-95. 16) Schmidt, N.J., and Lennette, E.H., 1962. Gel double diffusion studies with group B and group A, type 9 coxsackie viruses. II. Serologic diagnosis of coxsackie virus infections by the gel double diffusion technique. J. Immunol. 89: 96-105. 17) Tokuda, M., 1976, Studies on the casative agents of hand, foot and mouth disease p.131-144, In Virusgaku no shinten (Progress in Virology), Proceedings of the 16th symposium of the Institute of virus research, Kyoto university, 1976 (in Japanese). Requests for reprints should be addressed to Dr. Akio Hagiwara, Department of Enteroviruses, National Institute of Health, Musashimurayama, Tokyo 190-12, Japan,
Common
Antigen between Coxsackievirus A 16 and Enterovirus 71
Akio HAGIWARA, Isamu
TAGAYA, and
Tetsuo
YONEYAMA
Departmentof Enteroviruses,National Institute of Health, Tokyo (Received for publication, September 20, 1977)
Abstract Cross immunofluorescence revealed that coxsackievirus A 16 (CA 16) shared a common antigen with enterovirus 71 (E 71). The cross reactivity of these two serotypes was also examined by complement fixation test with purified virus preparations fractionated by sucrose density gradient centrifugation and two peaks of antigenicity were detected, one being type-specific and the other cross-reacting. The common antigen was heat-stable and attributable to empty capsids. Immunodiffusion also revealed the common antigen. Infants without antibody to E 71 developed complement fixing and precipitin antibody to E 71 after recovery from hand, foot and mouth disease caused by CA 16.
A widespread epidemic of hand, foot and mouth disease (HFMD) took place in 1973 in Japan and the causative agent was identified as E 71 (8). While we had been attempting to identify viruses isolated from patients in this epidemic, we noticed that virus isolates had a common antigen with CA 16 detectable by immunofluorescence. There was no neutralizability between these isolates and CA 16, and the isolates were finally identified as E 71. Further studies were carried out to clarify the antigenic relationship between these two serotypes and the results indicated that they share a common antigen attributable to empty capsids. MATERIALS
AND
METHODS
Cells. A continuous line of cynomolgus monkey cells established in our laboratory which was designated as CMK was used in most experiments. CMK cells were maintained in Eagle's minimal essential medium (Nissui Seiyaku Co.) with 10% bovine serum, 0.15% sodium bicarbonate and antibiotics. Primary cultures of cynomolgus monkey kidney cells (MK) were also used for virus preparation, which were grown in Earle's balanced salt solution with 0.5% lactalbumin hydrolysate and 2% bovine serum and antibiotics. Infected cells were maintained with serum-free medium. Viruses. Nagoya strain, a representative of E 71 isolates in 1973, was used most extensively. The prototype BrCr strain of E 71 was also used. The passage history of these two strains appeared elsewhere (8). The prototype G-10 strain of CA 16, originally received from CDC, Atlanta, USA, was passed from a homogenate of infected suckling mouse torsos to MK cells and then passed through CMK cells 81
82
A. HAGIWARA
12
to
14
times.
No
antigenic
change
was
ET AL
observed
during
the
monkey
kidney
cell
passages. Monolayers (ca. 0.1 maximum. for
PFU/cell) The
5 min
centrated on
a
of
cells
were
infected
with
an
and the cultures were harvested harvest was frozen and thawed
and
then
with
polyethylene
cushion
rotor.
CMK
of
centrifuged
CsCl
at glycol
(density
Band-formed
3,000
1.43)
virus
was
rpm
and at
10
min.
The
for
by
and
albino
supernate
through
rpm
purified
diluted
virus
cytopathic effect reached a times, sonicated at 10 MHz
centrifuged
40,000
further
when five
for
then
appropriately
90
CsCl
min
in
was
a Spinco
centrifugation
con-
(density
in
a
1.31) SW
CsCl
50.1
density
gradient. Immune G-10 each
of
crude
virus
virus
given
suspension
second mals
were
with
the
J.
immune
7 days
G-10 This
71
was
labeled
Sucrose the
top
pH 7.2 Fractions
a
linear
(12).
were
of put
a moistened
and purified
of the
injection. E
71
mixture
USA.
also
used
as
described
procedure
appeared G-10
16
in
and
a of
Ten
Purified
gradient
(15
was
to
carried
concentrated ml
virus
40%
of
out by
w/v)
days
serum the
later
the
The
ani-
prepared
courtesy fluid
WHO
of
Dr.
prepared
in
reference
horse
experiments. (8).
previous CA
(0.5 in
melted
1%
by
box
at
a
16
publication or
to
1.0
Nagoya
Noble
room
agar
ml)
veronal
(7). strain
in in
rpm
of
3 mm diameter the reagents
layered
density buffered
were cut were added,
on
saline
for
in
a CsCl
veronal
was
buffered
microtechnique
centrifugation
(10 •~•@ 12 cm). Wells of neighboring wells. After plastic
30
of the
isothiocyanate.
This were
The
amounts
ascitic
elsewhere
strain
days
suspension.
by
some
in
30
monkey
mice
CA
performed
with
and
available
of
5 ml
injection
equal
virus
with of
preparation.
of
immune
immune
strain
were
the
made
An
G-10
of
An
was
virus
(Difco),
2 ml
immunized injections
a booster
in a Spinco SW 50.1 rotor at 40,000 dropwise from the bottom of the tube.
fixation.
on a glass plate 5 mm between
the
were
subcutaneous
adjuvant
centrifugation.
sucrose
Antigens
in
of
fluorescein
gradient
Immunodiffusion. placed tance
last
prepared
and centrifuged were collected
Complement tray
with
density
of
The globulin
4 ml
with
strain
was
Immunofluorescence.
E
the
rabbits 4
days of
complete
prototype
against
immune
with
California,
Neutralization.
Monkey
injections
strain
the
given
alternate
intravenously
after
BrCr
with
serum
two
given
Schmidt,
laboratory
on
Freund's
was
bled
were
intramuscularly
prototype
Nathalie
given
and
injection
Monkeys
preparation
were
was
monkeys
strains.
Rabbits shot
our
Cynomolgus
Nagoya
the
later. first
sera.
and
2hr
a
at
at 5 C.
disposable gradient. saline with the
was a displates
temperature.
RESULTS
CrossImmunofluorescence betweenCA 16 and E 71 CMK cells infected with Nagoya strain were examined with labeled globulin against G-10 strain. Specific antigens were detected in the cytoplasm of infected cells (Fig. la). The specificity of the reaction was confirmed by inhibition of the staining with type-specific non-labeled immune serum as well as negative staining of non-infected cells and cells infected with the other coxsackievirus A or B which can
COMMON
ANTIGEN
BETWEEN CA
16 AND
E 71
83
a
b
Fig. I a. lmmunofluorescent staining of Nagoya strain-infected cells with an immune monkey globulin prepared with G-10 strain of CA 16. Fig. lb. I mmunofluoreseent staining of G-10 strain-infected cells with an immune monkey globulin prepared with Nagoya strain of 71.
grow in CMK cells and with several echovirus serotypes. The staining titer of this globulin for specific bright fluorescence was 1:4, while it was I :8 in CMK cells infected with the homologous virus. When CMK cells infected with the prototype BrCr of E 71 were examined, the same staining titer of 1:4 was obtained. CMK cells infected with G-10 strain could also be stained with the labeled globulin against Nagoya strain (Fig. lb), but in this case the staining titer was 1:1 (undiluted), while the homologous staining titer was 1:8. Twenty isolates of E71 in 1973 sent from various laboratories were also positive when tested for staining with the labeled globulin against G-10 strain. CrossReactionbetweenCA 16 and E 71 by ComplementFixation Preliminary experiments by use of an immune mouse ascitic fluid prepared with
84
A. HAGIWARA
ET AL
G-10 strain of CA 16 suggested that Nagoya strain shared a common complementfixing (CF) antigen with CA 16. The CF reactivity of the fractions of G-10 or Nagoya strain obtained by centrifugation in a sucrose density gradient was examined with the immune rabbit sera prepared with G-10 or Nagoya strain. As shown in Fig. 2a, two peaks of CF reactivity were shown when fractions of G-10 strain were examined with the homologous antiserum, which corresponded to the visible C and D bands reported with coxsackie- and polioviruses (6, 13). On the contrary, only a
b
Fig.
2. G-10
Complement-fixing strain
obtained
centrifugation
against
rologous sera. 30
activity by the
(anti-Nagoya a.
Native
min. •œ,
CF
rabbit
immune
against
an
sucrose
virus.
b.
Virus
titer
and
heated
CF rabbit
of
gradient
rabbit
against
serum; •›,
anti-Nagoya
fractions
homologous strain)
antigen
of density
hete-
immune at
56
C for
a homologous antigen immune
titer serum.
COMMON
ANTIGEN
BETWEEN
CA
16 AND
85
E 71
one peak, corresponding to the C band, was observed when the same fractions were examined with the anti-Nagoya serum. When the virus preparation was heated at 56 C for 30 min before centrifugation, the D band and the corresponding CF peak detectable with the homologous immune serum disappeared, while the fractions corresponding to the C band still showed a high CF reactivity against both the homologous and heterologous immune sera (Fig. 2b). A similar pattern of CF reactivity was shown with fractions obtained by centrifugation of Nagoya strain against the homologous and heterologous immune sera (data not shown). It is thus shown that empty capsids of CA 16 and E 71 share a common antigen reactive in the complement fixation test. Table
1.
Antibody
responses
of infants
after
the
primary
infection
with
CA
16
The antibody response of young children after the primary infection with CA 16 corroborated the above observations. In 1975 there observed some outbreaks of HFMD in Japan and viruses isolated from patients were identified as CA 16. We could examine paired sera of two patients born after 1973. As shown in Table 1, these children showed rises of both neutralizing and CF antibodies against CA 16 after recovery. When these sera were examined with E 71, only CF antibody was shown in the convalescent sera. Similar findings were also reported by Tokuda (17). CommonAntigenbetweenCA 16 and E 71 as Shownby Immunodiffusion Monkey immune sera prepared with G-10, Nagoya and BrCr strains were used, whose homotypic neutralizing antibody titered 1:500, 1:1,100 and 1:4,500 respectively. Paired sera of one of the HFMD patients used in the preceding experiment were also included. The native antigen was purified and concentrated by centrifugation in a CsCl solution about 1,000-fold by infectivity. A heated antigen was prepared by heating the native antigen at 60 C for 20 min. As shown in Fig. 3a, the convalescent serum of a patient showed the type-specific band with the native G-10 antigen and also the group-specific precipitin band with the native antigen of Nagoya and BrCr strains. This band appeared to coalesce with the band formed
86
A. HAGIWARA
ET Al,
a
b
c
Fig. 3. Cross immunodiffusion between CA 16 and E 71. Contents of holes: 1 =acute-phase serum from a CA 16 patient, 2 =Nagoya immune monkey serum, 3 = convalescent-phase serum from a CA 16 patient, 4 =G-10 immune monkey serum, 5= BrCr immune monkey serum (given by Dr. N.J. Schmidt), 6=native Nagoya strain, 7 =heated Nagoya strain, 8= native G-10 strain, 9= heated G-10 strain, 10= native BrCr strain.
between this serum and the native G-10 strain, but the continuity of the groupspecific band was not necessarily clear due to the overlapping of the type-specific and group-specific bands between the serum and the homologous antigen. When the heated G-10 antigen was used, the precipitin band due to the common antigen between the two serotypes was clearly observed as shown in Fig. 3b. Another experiment shown in Fig. 3c indicated that the band formed between the heated Nagoya antigen and the convalescent serum of the patient coalesced with those formed between the same antigen and the homologous anti-E 71 monkey sera as well as the monkey immune serum against CA 16 (G-10). The type-specific band formed between the native Nagoya virus and the homologous immune monkey sera crossed with the former group-specific precipitin band. DISCUSSION It has been established that the main etiologic agent of HFMD is CA 16. Recently two big epidemics of HFMD took place in Japan: one in 1969-70 was caused by CA 16, but the other in 1973 was proved to be due to E 71 infection as reported previously (8). During the course of identifying virus isolates, which later proved to be E 71, it was shown that they had a common antigen with CA 16 detectable by immunofluorescence. Although these two serotypes had no cross neutralizability, it was considered necessary to clarify the antigenic relationship between the
COMMON
ANTIGEN
BETWEEN
CA
16 AND
E 71
87
two serotypes because they brought about the same clinical symptoms. The results presented here have indicated that these two serotypes share common antigenicity attributable to empty capsids, which can be detected by immunofluorescence, complement fixation and immunodiffusion. Group antigen was demonstrated among coxsackievirus group B serotypes and group A type 9 by immunodiffusion (15, 16). Balayan et al studied the precipitin test with echovirus types 1, 6, 8, 13 and 19 as well as CA 7 and 9, and only CA 7 and 9 showed two precipitin lines with the respective standard monkey immune serum, one of them corresponding to the common antigen and the second to the type-specific antigen (1). Echovirus types 1 and 8 are antigenically closely related by cross neutralization tests and share common precipitin antigen(s) besides respective type-specific antigen (14). Conant et al studied echovirus 4 and other enteroviruses by gel precipitation and found that a serologic variant of echovirus 4, Shropshire and the prototype strain Pesascek shared a common precipitin antigen as well as type-specific antigens, which, however, did not appear to be completely identical, but with some strain specificity (3). From the results with coxsackievirus group B type 6 and echovirus 14, they suggested the existence of a group antigen shared by human enteroviruses, but the mutual relationship of their "B" lines was not clarified . With three types of poliovirus the different antigenicity of empty capsid from complete virion has been shown by complement fixation and immunodiffusion, but both antigens were type specific with immune animal sera (10, 11). Some human sera with monotypic neutralizing antibody reacted with heterotypic "C" or "H" antigens by complement-fixation, but other sera did not (10). Hinuma and Hummeler (9) reported that immunofluorescence of poliovirus was type-specific, although the antigens responsible for immunofluorescence were proved to be "H". Among coxsackievirus B serotypes French et al (5) reported that there observed some heterotypic and nonspecific staining (diffuse 1-2 + staining) at low serum dilutions by indirect immunofluorescence, but the immune reagents could be diluted to a point where they gave no heterotypic reactivity, but still showed characteristic homotypic staining. By direct immunofluorescence we observed that CMK cells infected with E 71 could be stained specifically with a labeled immune globulin against CA 16. As shown in Fig. 1a, brightly staining aggregates of viral antigen in the cytoplasm of infected cells were observed, and the staining titer of this globulin for 3-4 + staining was 1 :8 for CA 16-infected cells and 1:4 for E 71-infected cells. On the contrary, a labeled immune globulin prepared with Nagoya strain was more type-specific, the homologous staining titer being 1 :8, while the heterotypic titer was 1:1. In this case, the heterotypic staining was also specific with bright aggregates of antigen in the cytoplasm (Fig. 1b). The results obtained in the present experiments seem to indicate that the relationship between CA 16 and E 71 may be closer than among three types of poliovirus or among coxsackievirus group B and group A type 9. Classification of enteroviruses is based on neutralization, but three types of poliovirus are classified as one subgroup, while echovirus 1 and 8, which are related even by neutralization, are still grouped as independent serotypes (4). Some cross reactivity among several cox-
88
A. HAGIWARA
ET AL
sackievirus A serotypes has not been fully studied (2). Due to various biological and serological properties it may be reasonable to speculate that three serotypes of poliovirus might have evolved from the same root. In this connection CA 16 and E 71 may form another subgroup among enteroviruses. It may be of interest to study other enterovirus serotypes with an attempt of subgrouping some of them on the basis of common antigen as well as by the grade of homology of nucleic acids. We acknowledgethe courtesyof Drs. M. Tokuda, Institutefor VirusResearch,KyotoUniversity,and C. Miwa,GifuPrefecturalInstituteof PublicHealth,for providingus the pairedseraof HFMDpatients. REFERENCES
1)
Balayan, M.S., Belyaeva, A.P., and Seibel, V.B. 1963. Use of the precipitin test in the diagnosis of infections caused by ECHO and Coxsackie viruses. Acta Virol. 7: 241-249. 2) Committee on enteroviruses. 1962. Classification of human enteroviruses. Virol. 16: 501-504. 3) Conant, R.M., Barron, A.L., and Milgrom, F. 1966. Gel precipitation with echovirus 4 and other enteroviruses. Proc Soc. Exp. Biol. Med. 148: 203-207. 4) Fenner, F. 1976. Classification and nomenclature of viruses; Picornaviridae. Intervirology 7: 39. 5) French, M.L.V., Schmidt, N.J., Emmons, R.W., and Lennette, E.H. 1972. Immunofluorescence staining of group B coxsackievirus. Appl. Microbiol. 23: 54-61. 6) Frommhagens, L. 1965. The separation and physicochemical properties of the C and D antigens of coxsackievirus. J. Immunol. 95: 818-822. 7) Hagiwara, A., and Kitahara, T. 1974. Enhancing effect of green monkey kidney cell extract on the transformation of mouse cells by SV40. Int. J. Cancer 14: 26-31. 8) Hagiwara, A., Tagaya, I., and Yoneyama, T. 1978. Epidemic of hand, foot and mouth disease associated with enterovirus 71 infection. Intervirology 9: 60-63. 9) Hinuma, Y., and Hummeler, K. 1961. Studies on the complement-fixing antigens of poliomyelitis. III. Intracellular development of antigen. J. Immunol. 87: 367-375. 10) Hummeler, K., and Hamparian, V.V. 1958. Studies on the complement-fixing antigens of poliomyelitis. I. Demonstration of type and group specific antigens in native and heated viral preparations. J. Immunol. 81: 499-505. 11) Le Bouvier, G.L. 1959. Poliovirus D and C antigens: Their differentiation and measurement by precipitation in agar., Brit. J. Exp. Pathol. 40: 452-462. 12) Lennette, E.H. 1969. Complement fixation test. p. 52-58. In Diagnostic Procedures for viral and Rickttsial Infections, 4th ed, American Public Health Association, Inc. 13) Mayer, M.M., Rapp, H.J., Roizman, B., Klein, S.W., Crowan, K.M., Kukens, D., Schwerdt, F.L., and Charney, J., 1955. The purification of poliomyelitis virus as studied by complement fixation. J. Immunol. 78: 435-455. 14) Middleton, G.K., Jr., Cramblett, H.G., Moffet, H.L., Black, J.P., and Shulenberger, H. 1964. Micro diffusion precipitin tests for enteroviruses and influenza B virus. J. Bacteriol. 87: 11711176. 15) Schmidt, N.J., and Lennette, E.H. 1962. Gel double diffusion studies with group B and group A, type 9 coxsackie viruses. I. The technique and reactions obtained with hyperimmune animal sera and human sera. J. Immunol. 89: 85-95. 16) Schmidt, N.J., and Lennette, E.H., 1962. Gel double diffusion studies with group B and group A, type 9 coxsackie viruses. II. Serologic diagnosis of coxsackie virus infections by the gel double diffusion technique. J. Immunol. 89: 96-105. 17) Tokuda, M., 1976, Studies on the casative agents of hand, foot and mouth disease p.131-144, In Virusgaku no shinten (Progress in Virology), Proceedings of the 16th symposium of the Institute of virus research, Kyoto university, 1976 (in Japanese). Requests for reprints should be addressed to Dr. Akio Hagiwara, Department of Enteroviruses, National Institute of Health, Musashimurayama, Tokyo 190-12, Japan,
