Journal
of General Virology (1998), 79, 2255–2263. Printed in Great Britain
...................................................................................................................................................................................................................................................................................
Antiviral activity of bovine collectins against rotaviruses
Patrick C. Reading,1 Uffe Holmskov2 and E. Margot Anders1
1
2
Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria 3052, Australia
Department of Medical Microbiology, Institute of Medical Biology, University of Odense, DK-5000 Odense C, Denmark
The antiviral activity against rotaviruses of three
bovine collectins, conglutinin, collectin-43 (CL-43)
and bovine SP-D, was examined. As shown by ELISA
and Western blot, all three collectins bound to the
Nebraska calf diarrhoea virus bovine strain of
rotavirus, and specifically to the VP7 glycoprotein.
Inhibition by mannose or EDTA confirmed that
binding was mediated through the lectin domains of
the collectins. Binding resulted in haemagglutination inhibition and neutralization of rotavirus infectivity, CL-43 displaying the highest activity in
both types of assay. In contrast, conglutinin was the
most potent of the three collectins against influenza
Introduction
Rotaviruses are the main cause of acute viral gastroenteritis
in infants and young animals, but subclinical infections are also
common. Host factors that influence susceptibility to symptomatic rotavirus infection are not fully understood. Some
instances of subclinical infection may reflect partial crossprotection afforded by immunity to a related strain of rotavirus
previously encountered. However, variation in clinical outcome also occurs in the immunologically naive, indicating the
existence of non-immune or innate immune factors affecting
susceptibility, some of which may be age-dependent (Tzipori et
al., 1981 ; Bridger, 1994). These factors may include a decreased
susceptibility of the maturing enterocyte to rotavirus infection
(Riepenhoff-Talty et al., 1982) and inhibition of rotavirus
replication by intestinal mucins (Chen et al., 1993).
In recent years there has been increasing interest in the role
of collectins in innate host defence against microbial infection
(for review, see Holmskov et al., 1994 ; Hoppe & Reid, 1994 ;
Ezekowitz et al., 1996). The collectins are a family of oligomeric
proteins composed of trimeric subunits that contain collagenAuthor for correspondence : E. Margot Anders.
Fax ­61 3 9347 1540.
e-mail m.anders!microbiology.unimelb.edu.au
virus A/HKx31. Neutralization of rotaviruses by the
lectins was dependent on glycosylation of VP7.
Furthermore, rotaviruses adapted to growth in
Madin–Darby bovine kidney cells, and thus bearing
carbohydrate of bovine origin, remained sensitive
to neutralization, although slightly less so than virus
stocks propagated in the monkey kidney cell line
MA104. These findings provide the first description
of antiviral activity of collectins against a nonenveloped virus and may indicate a potential role
for collectins in host defence against bovine rotavirus infection.
like sequences linked to Ca#+-dependent (C-type) lectin
domains (Thiel & Reid, 1989). Members of this family include
serum mannose-binding lectin (MBL ; also termed mannosebinding protein, MBP) ; the lung surfactant proteins A (SP-A)
and D (SP-D) ; and two additional plasma collectins, conglutinin
and collectin-43 (CL-43), which are found only in cattle and
other Bovidae. CL-43 differs from the other collectins in that it
does not appear to form higher oligomers of the trimeric
subunit (Holmskov et al., 1995). The collectins all bind to
mannan and mannose-containing glycans but display distinct
differences in their specificity for particular monosaccharides
and oligosaccharides. They have been shown to bind to a
variety of Gram-positive and Gram-negative micro-organisms,
yeasts and parasites, and to act as opsonins, promoting
phagocytosis probably through interaction with specific
receptors on phagocytic cells.
Collectins also bind to the glycoproteins of a number of
enveloped viruses including human immunodeficiency virus
(Ezekowitz et al., 1989 ; Andersen et al., 1991), herpes simplex
virus (Fischer et al., 1994) and influenza virus (Anders et al.,
1990 ; Malhotra et al., 1994 ; Reading et al., 1997). Binding of
MBL, SP-D and conglutinin to influenza virus has been shown
to mediate a range of antiviral activities in vitro including virus
aggregation, neutralization of virus infectivity and enhancement of virus interaction with neutrophils (Anders et al., 1990,
0001-5394 # 1998 SGM
CCFF
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P. C. Reading, U. Holmskov and E. M. Anders
1994 ; Hartshorn et al., 1993 a, b, 1994 ; Reading et al., 1997). A
recent study correlating sensitivity to collectins with the
relative ability of influenza virus strains to replicate in mouse
lung pointed to SP-D as an important component of host
defence of the lung against influenza virus infection in mice
(Reading et al., 1997). However, not all collectin–virus
interactions are inhibitory. Binding of MBL or conglutinin to
herpes simplex virus type 2 did not neutralize virus infectivity
and, when administered to mice before infection with the virus,
these collectins enhanced rather than inhibited virus replication
in the liver (Fischer et al., 1994). As yet no antiviral activity has
been described for CL-43, nor for conglutinin against a bovine
virus.
Rotaviruses are unusual amongst non-enveloped viruses in
having a glycoprotein as one of their two outer capsid
proteins. This glycoprotein, VP7, forms the smooth surface of
the virion from which the other outer capsid protein, VP4,
protrudes as spikes. VP4 is the cell attachment protein and the
viral haemagglutinin (Kalica et al., 1983 ; Ludert et al., 1996), but
antibodies directed towards either VP4 or VP7 can neutralize
virus infectivity (Matsui et al., 1989 ; Ruggeri & Greenberg,
1991). Both VP4 and VP7 have been implicated in virulence
(Burke & Desselberger, 1996). The glycans of VP7 are all of the
high-mannose type, consistent with trimming of oligosaccharide and maturation of rotavirus particles exclusively in
the endoplasmic reticulum (Kouvelos et al., 1984 ; Kabcenell &
Atkinson, 1985). Since collectins show reactivity towards
high-mannose structures, it was of interest to determine
whether these molecules show any antiviral activity against
rotaviruses. Conglutinin has been detected in bovine colostrum
as well as in plasma (Ingram & Mitchell, 1970) and SP-D,
originally thought to be confined to lung surfactant, has
recently been shown to be present at other sites including the
gastrointestinal tract (Fisher & Mason, 1995). Both of these
collectins may thus have the opportunity of encountering
rotavirus particles during natural infection of calves. In this
study, we have examined the antiviral activity of the bovine
collectins conglutinin, CL-43 and bovine SP-D (bSP-D) against
bovine strains of rotavirus.
Methods
+ Cells and viruses. The rotavirus strains used in this study, and the
number and location of potential glycosylation sites on the VP7
glycoprotein of these viruses, are listed in Table 1. Bovine rotavirus
strains Nebraska calf diarrhoea virus (NCDV, Lincoln strain), B223, B-11
and B-60 (Mebus et al., 1971 ; Woode et al., 1983 ; Huang et al., 1992), the
simian rotavirus SA11 (Malherbe & Strickland-Cholmley, 1967), and a
mutant of SA11, SA11 clone 28 (Estes et al., 1982), were provided by Ian
Holmes, Department of Microbiology and Immunology, University of
Melbourne.
MA104 monkey kidney cells grown in RPMI 1640 supplemented
with 2 mM glutamine, 2 mM pyruvate, 30 µg}ml gentamicin and 10 %
foetal calf serum were used for the cultivation of rotaviruses. Prior to
infection, cell monolayers were washed with serum-free medium and
virus was treated with porcine trypsin type IX (10 µg}ml ; Sigma) for
CCFG
30 min at 37 °C. Activated virus was adsorbed to cells for 60 min at
37 °C, the inoculum was removed, and the cells were cultured in serumfree medium containing 0±5 µg}ml trypsin. When the cytopathic effect
was almost complete, the infected cells were frozen and thawed twice and
cell debris was removed by low-speed centrifugation. The supernatant
containing rotavirus was stored at ®70 °C until use. Rotavirus strains B60 and B-11 were adapted to growth in Madin–Darby bovine kidney
(MDBK) cells by ten sequential passages in these cells, following a
protocol similar to that described above.
NCDV particles were purified from virus-infected MA104 cell
cultures. Infected monolayers were subjected to two rounds of freeze–
thawing followed by treatment with 0±25 % (v}v) Arklone P (ICI) and
low-speed centrifugation to remove cell debris. Virus was concentrated
by ultracentifugation at 100 000 g for 75 min, and the pellet was then
centrifuged through 30–60 % glycerol gradients at 100 000 g for 120
min. The virus band corresponding to triple-shelled particles was
collected, diluted and concentrated again by ultracentrifugation. The
purified virus was resuspended in TBS (0±05 M Tris–HCl, 0±15 M NaCl
pH 7±2) containing 2 mM CaCl and frozen at ®70 °C until required.
#
Influenza virus strain A}HKx31 (H3N2) is a laboratory-derived
reassortant of A}Aichi}2}68 (H3N2)¬A}PR}8}34 (H1N1) in which
the genes for the haemagglutinin and neuraminidase glycoproteins are
derived from A}Aichi}2}68 (Kilbourne, 1969). HKx31 was grown in the
allantoic cavity of 10-day embryonated hens’ eggs by standard
procedures and purified by differential centrifugation and passage
through sucrose gradients as described (Anders et al., 1990). Protein
concentrations of purified viruses and viral glycoproteins were determined using the BCA protein assay kit (Pierce).
+ Lectins and antibodies. Purification of conglutinin and CL-43
from bovine serum (Holmskov et al., 1993) and bSP-D from bovine lung
lavage (Holmskov et al., 1995) were carried out as previously described.
Antisera to the purified proteins were raised in rabbits and the specificity
confirmed by Western blotting. Monoclonal antibody (MAb) A-3,
specific for the nucleoprotein of type A influenza viruses, was provided
by Nancy Cox, Influenza Branch, Centers for Disease Control and
Prevention, Atlanta, Georgia, USA. MAb C4}B3 was a gift from Barbara
Coulson, Department of Microbiology and Immunology, University of
Melbourne ; this MAb was raised against the VP7 of simian rotavirus
strain SA11 but cross-reacts with other rotavirus strains in ELISA (Sonza
et al., 1984) and Western blot (Barbara Coulson, personal communication).
Rabbit antiserum against rotavirus strain SA11 was prepared by Ieva
Lazdins, Department of Microbiology and Immunology, University of
Melbourne.
+ ELISA to detect binding of collectins to virus. Wells of flexible
polyvinyl microtitre trays were coated with a series of concentrations of
purified viruses in 50 µl TBS containing 0±1 % NaN , blocked for 3 h with
$
10 mg}ml BSA, and washed with TBS containing 0±05 % Tween 20 (TBST). The wells were then incubated overnight with 50 µl of conglutinin,
CL-43 or bSP-D in TBS-T containing 5 mg}ml BSA and either 50 mM
CaCl (BSA –TBS-T–Ca#+) or 5 mM EDTA (BSA –TBS-T–EDTA), and
#
&
&
then washed. Binding of conglutinin, CL-43 or bSP-D was detected by
the addition of the appropriate rabbit antiserum (1}200 dilution in
BSA –TBS-T–Ca#+) for 3 h, followed by the addition of HRP-conjugated
&
swine anti-rabbit immunoglobulin (DAKO) and substrate [2, 2«-azinobis(3-ethyl-benzthiazoline-6-sulphonic acid) ; 0±2 mM in 50 mM citrate
buffer pH 4±0, containing 0±004 % H O ].
# #
+ Western blot analysis of collectin binding to rotavirus
proteins. Proteins of purified NCDV were separated by SDS–PAGE on
a 10 % gel under non-reducing conditions and transferred to nitrocellulose
(Schleicher and Schuell). The membrane was blocked with TBS containing
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Bovine collectins and rotavirus
Table 1. Number and location of potential N-linked glycosylation sites on VP7 of
different rotavirus strains
Glycosylation at amino acid residue :*
Rotavirus strain
Serotype†
69–71
NCDV
B-60
B-11
B223
SA11
SA11 clone 28‡
G6P[1]
G6P[5]
G10P[11]
G10P[11]
G3P[2]
G3P[2]
­
­
­
­
146–148
­
238–240
318–320
Total
­
­
­
­
­
2
3
2
2
1
0†
* Potential N-linked glycosylation sites (Asn-X-Ser}Thr) (Kornfeld & Kornfeld, 1985) on the VP7
glycoprotein as obtained from published sequences : (Both et al., 1983 ; Glass et al., 1985 ; Xu et al., 1991 ;
Huang et al., 1992).
† All serotypes from Estes (1996) except B-11 and B-60 (personal communication : Jin-an Huang, St
Vincent’s Institute of Medical Research, Fitzroy, Victoria, Australia).
‡ SA11 clone 28 is a mutant of SA11 which has been shown to bear a non-glycosylated VP7 (Estes et al.,
1982).
5 mg}ml BSA (BSA –TBS). Individual tracks were probed with con&
glutinin, CL-43 or bSP-D (5 µg}ml), rabbit antiserum to rotavirus strain
SA11 (1}1000) or MAb C4}B3 to rotavirus VP7 (1}500). The diluent for
this and subsequent binding steps was BSA –TBS supplemented with
&
20 mM CaCl (BSA –TBS–Ca#+). Following overnight incubation,
#
&
membranes were washed five times in TBS-T supplemented with 20 mM
CaCl (TBS-T–Ca#+). To detect bound lectins, rabbit antiserum to
#
conglutinin, CL-43 or bSP-D (1}300) was applied to the membranes for
3 h. After the membranes were washed, mouse and rabbit antibodies
were detected with HRP-conjugated rabbit anti-mouse immunoglobulin
or swine anti-rabbit immunoglobulin (DAKO), respectively, followed by
incubation with substrate (3,3« diaminobenzidine hydrochloride ;
0±69 mM in TBS containing 0±0075 % H O ). In some experiments lectins
# #
were applied to the membrane in BSA –TBS–Ca#+ supplemented with
&
100 mM -mannose or in BSA –TBS containing 5 mM EDTA.
&
+ Haemagglutination inhibition tests. Haemagglutination inhibition (HI) tests were performed by standard procedures (Palmer et al.,
1975) in 96-well plates using four haemagglutinating doses of virus and
1 % (v}v) chicken or guinea-pig erythrocytes for influenza virus and
rotavirus, respectively. Complement fixation test (CFT) buffer (Oxoid :
barbitone-buffered saline pH 7±2, 0±25 mM CaCl , 1±8 mM MgCl ) was
#
#
used as the diluent.
+ Virus neutralization assays. Neutralization of rotavirus or
influenza virus infectivity was measured by fluorescent focus reduction
assays in monolayers of MA104 or Madin–Darby canine kidney cells,
respectively. Dilutions of conglutinin, CL-43 or bSP-D were prepared in
CFT diluent and mixed with virus in a total volume of 100 µl. Rotavirus
had been preactivated with 10 µg}ml porcine trypsin before use. The
dose of virus used was that which gave approximately 50 fluorescent foci
per low-power field (see below) after incubation in CFT alone. The
mixtures were incubated at 37 °C for 30 min, and then 50 µl was
inoculated onto confluent cell monolayers in 96-well plates (Nunc) that
had been washed with RPMI 1640 supplemented with 30 µg}ml
gentamicin. After adsorption of the virus for 45 min at 37 °C, 5 % CO ,
#
the inoculum was removed, and 100 µl of the same medium was added to
each well. Plates were incubated a further 7–8 h for influenza virus, or
13–14 h for rotavirus, and then the cells were fixed with 80 % (v}v)
acetone. Wells were stained for influenza virus by incubation with 50 µl
1}1000 dilution of the anti-NP MAb A-3 in TBS, followed by fluoresceinconjugated sheep anti-mouse immunoglobulin (Silenus). Rotavirusinfected cells were detected by incubation with 1}1000 dilution of rabbit
antiserum against rotavirus strain SA11, followed by fluoresceinconjugated sheep anti-rabbit immunoglobulin (Silenus). Plates were
viewed under ¬128 magnification and the total number of fluorescent
foci in four representative fields were counted and expressed as a
percentage of the number of foci in the corresponding area of control
wells infected with virus alone.
To test for inhibition of neutralization by sugars, lectin dilutions were
prepared in CFT buffer containing the sugar and held at room temperature
for 20 min before the addition of virus.
Results
Binding of conglutinin, CL-43 and bSP-D to bovine
rotavirus and influenza A virus
The ability of conglutinin, CL-43 and bSP-D to bind to the
bovine rotavirus NCDV was tested by ELISA. As a positive
control, binding of the lectins to the type A influenza virus
HKx31 was also examined. Fig. 1 shows the results of an assay
in which microtitre wells were coated with increasing concentrations of purified viruses, and the binding of a standard
concentration of lectin (5 or 1 µg}ml) was determined. All
three lectins bound to NCDV, although to a substantially
lower level than to HKx31 virus. The relative efficiency of
binding of the three lectins to a single virus could not be
determined since different antisera were used in developing
each of the assays.
Collectins have been shown to bind to both the haemagglutinin and the neuraminidase glycoproteins of influenza virus
(Anders et al., 1990 ; Hartley et al., 1992 ; Malhotra et al., 1994 ;
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P. C. Reading, U. Holmskov and E. M. Anders
Fig. 2. Western blot showing the binding of specific antibodies and bovine
collectins to NCDV. The proteins of NCDV were separated by SDS–PAGE
under non-reducing conditions on a 10 % gel, and transferred to a
nitrocellulose membrane. The membrane was incubated with polyclonal
antiserum raised against rotavirus strain SA11 (pAb), with MAb C4/B3
which is specific for VP7 (anti-VP7) or with 5 µg/ml of the collectins
conglutinin (K), CL-43 or bSP-D, as indicated. Bound collectins were
detected by incubation with specific antibodies. Bound antibodies were
then visualized by incubation with HRP-conjugated anti-immunoglobulins
and substrate as described under Methods. All binding steps were
performed in the presence of 20 mM CaCl2.
with erythrocyte receptors (Matsui et al., 1989). As shown in
Table 2, haemagglutination of guinea-pig erythrocytes by
NCDV was also inhibited by the three collectins, CL-43
displaying the highest activity. In contrast, against influenza
virus HKx31, conglutinin was more potent than CL-43. In all
cases, HI activity was inhibited by 50 mM -mannose, but not
by the control sugar -rhamnose.
Fig. 1. ELISA measuring binding of conglutinin, CL-43 and bSP-D to
rotavirus strain NCDV (+) and influenza virus strain HKx31 (E). Wells
were coated with increasing concentrations of purified viruses and lectins
were applied in 50 mM CaCl2. Lectins were used at a concentration of 5
and 1 µg/ml for NCDV and HKx31, respectively.
Reading et al., 1997). To identify the viral protein(s) on NCDV
to which these collectins bind, purified virus was analysed by
SDS–PAGE and Western blot. Of the several protein species
that could be detected with polyclonal antiserum, conglutinin,
CL-43 and bSP-D bound to a single species of approximately
35 kDa, corresponding in position to the band recognized by
a monoclonal antibody raised against the VP7 glycoprotein
(Fig. 2). Binding of the collectins was inhibited in the presence
of 5 mM EDTA or 100 mM -mannose (data not shown),
indicating that binding occurred through the lectin domain of
these proteins to oligosaccharide on the VP7 glycoprotein.
Inhibition of rotavirus and influenza virus-mediated
haemagglutination by bovine collectins
Although VP4 is the rotavirus haemagglutinin (Kalica et al.,
1983), antibodies to VP7 may also exhibit HI activity,
presumably through steric hindrance of the interaction of VP4
CCFI
Neutralization of bovine rotaviruses and influenza
virus by conglutinin, CL-43 and bSP-D
The infectivity of NCDV was neutralized by each of the
three collectins (Fig. 3 A). Virus neutralization was abolished
by the addition of -mannose to the collectins before the
addition of virus (final concentration of -mannose, 50 mM),
whereas the control sugar, -rhamnose, was without effect.
Virus infectivity was also restored if -mannose was added
after incubation of virus–collectin mixtures at 37 °C (data not
shown), indicating that collectin binding did not irreversibly
destroy virus infectivity. Neutralization of influenza virus
HKx31 by the three collectins is shown in Fig. 3 (B). As
observed for HI (Table 2), the relative potency of the lectins in
neutralizing NCDV and HKx31 virus differed, CL-43 being the
most active against NCDV and conglutinin the most active
against HKx31. HKx31 was neutralized by lower concentrations of each of the three lectins than were needed for
neutralization of NCDV, consistent with the higher level of
binding of the lectins to HKx31, as determined by ELISA
(Fig. 1).
Rotaviruses differ in the number and location of potential
N-linked glycosylation sites on the VP7 molecule (Table 1),
and it was of interest to determine the effect of different
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Bovine collectins and rotavirus
Table 2. Bovine collectins inhibit haemagglutination by rotavirus and influenza virus
HIC50*
Rotavirus NCDV
Diluent
CFT†
­50 mM -mannose
­50 mM -rhamnose
Influenza virus HKx31
Conglutinin
CL-43
bSP-D
Conglutinin
CL-43
bSP-D
5±0
50
5±0
0±6
50
1±2
5±0
50
5±0
0±3
25
0±3
0±6
25
1±2
5±0
50
5±0
* Minimum concentration of collectin (µg}ml) required to inhibit 4 haemagglutinating units of virus. Guineapig and chicken erythrocytes (1 %, v}v) were used for rotavirus and influenza virus respectively.
† Complement fixation test diluent (Oxoid ; barbitone-buffered saline pH 7±2, 0±25 mM CaCl , 1±8 mM
#
MgCl ).
#
tested, and the variant SA11 clone 28, which has a nonglycosylated VP7 (Estes et al., 1982), was resistant to
neutralization by the collectins.
Collectin sensitivity of MDBK cell-grown rotaviruses
Fig. 3. Neutralization of (A) NCDV and (B) HKx31 by conglutinin (+, *),
CL-43 (E, D) and bSP-D (_, ^). Collectins in CFT diluent alone (closed
symbols) or diluent supplemented with 50 mM mannose (open symbols)
were assessed for their ability to neutralize virus infectivity, as measured
by a fluorescent focus reduction assay. The infectivity of the viruses in CFT
(y) and CFT supplemented with 50 mM mannose (x) in the absence of
collectins is also shown. Neutralization assays conducted in the presence
of 50 mM rhamnose gave results essentially similar to those obtained in
the absence of sugars (data not shown). Experiments were performed on
three occasions with similar results.
The oligosaccharide structures on viral glycoproteins can
differ depending on the host cell in which the virus was grown
(Kornfeld & Kornfeld, 1985). For the experiments described
above, the rotaviruses had all been grown in MA104 cells, a
foetal rhesus monkey kidney cell line. To determine whether
the bovine collectins also recognize virus bearing oligosaccharide of bovine cell origin, an attempt was made to adapt the
bovine rotaviruses to growth in the MDBK cell line, and this
was achieved with strains B-11 and B-60. As shown in Fig. 5,
MDBK-grown viruses remained sensitive to neutralization by
the collectins, although less so than their MA104-grown
counterparts. Influenza virus grown in MDBK cells likewise
remained sensitive to the bovine collectins, but reproducibly
less so than egg-grown virus stocks (data not shown).
Discussion
patterns of glycosylation on collectin sensitivity of the viruses.
The bovine strains NCDV, B-11, and B223 with two, and B-60
with three, potential glycosylation sites were all sensitive to
neutralization by conglutinin, CL-43 and bSP-D (Fig. 4).
NCDV and B-60 were consistently more sensitive than B-11
and B223 in repeated assays, suggesting perhaps an important
contribution to collectin sensitivity by the carbohydrate at
residue 318–320. It must be noted, however, that actual usage
of all of the glycosylation sites in these strains has not yet been
established.
In contrast to the bovine strains, the simian rotavirus SA11,
which has a single potential glycosylation site on VP7, was
neutralized only at the highest concentrations of each collectin
This study has demonstrated antiviral activity of purified
bovine collectins against bovine rotaviruses in vitro. Binding of
conglutinin, CL-43 and bSP-D through their lectin domains to
the VP7 glycoprotein of rotavirus strain NCDV was shown by
Western blot, and the collectins displayed both HI and
neutralizing activity. Neutralization of rotavirus was dependent upon glycosylation of VP7. Furthermore, viruses adapted
to the MDBK cell line, and hence bearing carbohydrate of
bovine origin, remained sensitive to neutralization by these
bovine lectins. These findings provide the first description of
antiviral activity of collectins against a non-enveloped virus
and suggest a potential role for collectins in host defence
against rotavirus infection, as discussed further below.
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P. C. Reading, U. Holmskov and E. M. Anders
Fig. 4
Fig. 5
Fig. 4. Neutralization of different rotavirus strains by conglutinin, CL-43 and bSP-D using a fluorescent focus reduction assay.
Collectins were examined for their ability to neutralize the bovine rotavirus strains NCDV (_), B223 (^), B-11 (+) and B-60
(E), the simian rotavirus strain SA11 (y) and a variant of SA11, clone 28, which bears a non-glycosylated VP7 (x).
Experiments were performed on three occasions with similar results.
Fig. 5. Neutralization of MA104-grown (closed symbols) and MDBK-grown (open symbols) bovine rotaviruses by conglutinin,
CL-43 and bSP-D : B-60 (E, D) and B-11 (+, *). Neutralization was abrogated in the presence of 50 mM mannose (data
not shown). Experiments were performed on three occasions with similar results.
In examining the antiviral activities of conglutinin, CL-43
and bSP-D against rotaviruses, we used influenza virus strain
HKx31 for comparison. Influenza virus does not naturally
infect cattle, but is a virus for which the antiviral activities of
collectins are well characterized. Both conglutinin and SP-D
function as β inhibitors of influenza virus, binding to viral
carbohydrate and mediating HI and virus neutralization
(Anders et al., 1990 ; Hartley et al., 1992 ; Hartshorn et al., 1994 ;
Reading et al., 1997) as well as causing virus aggregation and
opsonizing the virus for interactions with neutrophils
(Hartshorn et al., 1993 a, 1994). The antiviral activity of CL-43
CCGA
had not been examined previously. As shown here, CL-43
displays HI and neutralizing activity against influenza virus
and thus represents a second β inhibitor in bovine serum. Since
CL-43 exists as single trimeric subunits (Holmskov et al., 1995),
subunit oligomerization is not obligatory for antiviral activity,
at least of this collectin.
The sensitivity of NCDV to the three collectins was lower
than that of influenza virus HKx31, as seen both in direct
binding by ELISA and in the concentration of collectins
required for virus neutralization. Factors contributing to this
difference will include both the overall level and type of
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Bovine collectins and rotavirus
glycosylation of the two viruses, and the extent to which the
oligosaccharide side chains are exposed on the virion and
available for collectin binding. NCDV and the other bovine
rotaviruses tested bear a single species of glycoprotein, VP7,
which carries high-mannose type carbohydrate attached at two
or three N-linked potential glycosylation sites (Table 1).
Influenza virus HKx31 carries seven N-linked potential glycosylation sites on its haemagglutinin (Ward & Dopheide,
1981) and six or seven on its neuraminidase (Colman & Ward,
1985), and the oligosaccharides include high-mannose as well
as complex types. It is not unexpected, therefore, that influenza
virus would present more binding sites for the collectins than
rotavirus, although the exact stoichiometry of binding is
unknown.
Rotaviruses also differed from influenza virus in their
pattern of reactivity with the three collectins. For all four
bovine rotavirus strains tested, CL-43 displayed higher
antiviral activity than conglutinin or bSP-D, whereas conglutinin was the most potent of the collectins against HKx31.
These differences in reactivity are consistent with welldocumented differences in fine specificity of these collectins.
All bind to mannose-containing glycans, but differ in their
affinities for particular monosaccharides and oligosaccharides,
presumably due to sequence differences within the carbohydrate recognition domains of each collectin (Holmskov et al.,
1994 ; Lim et al., 1994). The relative affinities of the different
collectins for a particular virus will thus depend on the
particular glycan structures present on the virus and their
accessibility to each collectin (Solı! s et al., 1994 ; Loveless et al.,
1995).
Neutralization of rotavirus by collectins may occur by a
number of mechanisms. Current evidence points to rotavirus
VP4 as the major cell attachment protein (Ludert et al., 1996) as
well as the viral haemagglutinin (Kalica et al., 1983). Haemagglutinin inhibition of rotavirus by collectins bound to VP7
most probably occurs through steric hindrance of access of
VP4 to sialylated erythrocyte receptors, and neutralization of
rotavirus by collectins may occur by a similar mechanism.
Alternatively, and by analogy with certain neutralizing
antibodies directed against VP7 (Ruggeri & Greenberg, 1991),
the bound collectins may interfere not with initial binding, but
with a subsequent step in viral entry which could involve
either VP4 or VP7 itself. In a recent study, Coulson et al. (1997)
have identified integrin ligand sequences on both VP4 and VP7
of rotaviruses, and provided evidence implicating these
sequences in the entry of rotaviruses into cells. Blocking of
rotavirus–integrin interactions would thus represent a possible
mechanism of neutralization by collectins.
Virus aggregation may also contribute to neutralization of
rotavirus by collectins, by reducing the number of infectious
units and perhaps also by interfering physically with virus
entry. Conglutinin and SP-D, which are large oligomeric
molecules, both show strong aggregation of influenza virus
(Hartshorn et al., 1993 a, 1994). Aggregation by CL-43, which
exists as single trimers, is less likely unless the individual
carbohydrate recognition domains on the one trimer can crosslink two virus particles.
The potential biological significance of collectin–rotavirus
interaction depends on whether collectins have access in vivo
to the site of rotavirus infection, i.e. the small intestine. The
following observations support this possibility.
SP-D was originally identified as a component of lung
surfactant produced by alveolar type II cells and non-ciliated
bronchiolar cells (Clara cells) in the respiratory tract (Crouch et
al., 1992). More recent studies have identified SP-D in other
body compartments, including the gastrointestinal tract. Thus
SP-D and SP-D mRNA were detected in both the salivary
glands and the small intestine of humans by immunohistochemical studies and RT–PCR, respectively (U.
Holmskov, unpublished observations). Expression of SP-D
mRNA was also detected in the stomach of rats, mice and
humans (Fisher & Mason, 1995 ; Motwani et al., 1995), and
surfactant-like particles containing a protein cross-reactive
with SP-D were identified within enterocytes and in the
intestinal lumen of rats (Eliakim et al., 1991). (In that study, SPD was referred to by its former name, CP4.) The intestinal
surfactant-like particles did not contain SP-A and so are
unlikely to represent swallowed lung surfactant.
Bovine conglutinin and CL-43 are plasma collectins synthesized in the liver. The presence of conglutinin in colostrum has
also been reported (Ingram & Mitchell, 1970), and immunochemical studies on sections of bovine tissues have also
detected conglutinin associated with follicular dendritic cells,
macrophages and endothelial cells (Holmskov et al., 1992). In a
study of 27 cows over 6 months old, the plasma concentrations
of conglutinin ranged from 3±5 to " 56 µg}ml (mean
29±0³12±1 µg}ml) and of CL-43 from 0±6 to 9±24 µg}ml
(mean 4±6³2±4 µg}ml) (U. Holmskov & I. Tornøe, unpublished observations), the higher levels of which may be
sufficient to neutralize or opsonize rotavirus if it entered the
bloodstream. Whilst rotavirus infection is generally confined
to the small intestine, the reported detection of rotaviral RNA
in blood and cerebrospinal fluid of children with convulsions
and gastroenteritis (Nishimura et al., 1993) suggests the
potential for viraemic spread. Plasma collectins such as
conglutinin and CL-43 in cattle, and serum MBL in humans and
other mammals, may thus conceivably play a role in preventing
viraemia. Furthermore, transudation of MBL into inflammatory
sites such as the rheumatoid joint (Malhotra et al., 1995) or the
influenza virus-infected lung (Reading et al., 1997) has been
described. Whether transudation of MBL, conglutinin or CL-43
into the intestinal lumen occurs during rotavirus infection has
yet to be determined.
There is considerable variation in the number and location
of potential glycosylation sites on the VP7 of different
rotavirus strains. Whilst the biological significance of these
glycosylation differences is not fully understood (Estes &
Cohen, 1989), studies with monoclonal antibody-selected
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P. C. Reading, U. Holmskov and E. M. Anders
escape mutants of rotaviruses have shown that glycosylation
can have marked effects on the antigenicity of VP7 (Caust et al.,
1987 ; Lazdins et al., 1995). If collectins play a role in innate host
defence against rotavirus infection, the level of glycosylation
of VP7 might be expected to affect the ability of rotavirus to
replicate in the gut, strains which are more highly glycosylated
being more sensitive to collectin-mediated neutralization or
opsonization. Studies examining virus replication and disease
in animal models using strains of rotavirus that differ only in
the degree of glycosylation of their VP7 molecules may thus
prove informative in evaluating the potential role of collectins
in host defence against rotaviruses.
This work was supported by the National Health and Medical
Research Council of Australia (Grant 940742 to E. M. A.) and the Benzon
Foundation (U. H.). We thank Mrs Ida Tornøe for skilled technical
assistance and Drs Ian Holmes and Barbara Coulson for stimulating
discussions.
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Received 17 December 1997 ; Accepted 21 April 1998
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