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Regular Articles |
From the Department of Neuropharmacology,*
The Scripps
Research Institute, La Jolla, California; and
Pharmadigm,
Salt Lake City, Utah
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Several avenues of research have implicated B cells as important to the interaction between the host and these viruses. First, antibodies seem to be critical to the control of these viral infections. Individuals genetically deficient in antibody production can resist most viruses but are susceptible to chronic enterovirus infections; although many of these are echoviral,3,4 CVB encephalitis also has been noted.5,6 Adoptive transfer of immune serum sometimes, but not always, ameliorates disease. Second, recent work suggests that B cells may be an important site of early CVB replication in vivo,7,8 raising the possibility that these cells may contribute to the virus' ability to establish or disseminate infection, and it is tempting to suggest that infection of these cells might contribute to the immunosuppression reported more than 20 years ago.9,10
We therefore sought to clarify the role of B cells in CVB pathogenesis and investigated (i) whether CVB3 infection of B lymphocytes is productive, (ii) the frequency of productively infected B and non-B splenocytes, (iii) the splenic distribution of CVB in mice lacking B lymphocytes (BcKO mice), (iv) the role of the murine coxsackievirus-adenovirus receptor (mCAR) in B cell infection, (v) the contribution of B cells to early replication and dissemination of CVB3, (vi) the susceptibility of BcKO mice to long-term enteroviral infection as seen in human agammaglobulinemia, and (vii) the efficacy of B and T cell transfer in control of long-term infection.
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Materials and Methods |
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A myocarditic strain of CVB3 (Nancy) was cloned and sequenced11 (GenBank accession number U57056), and plasmid pH3, encoding this genome, was kindly provided by Dr. Kirk Knowlton of the University of California (San Diego, CA). Transfection of HeLa cells with this plasmid yields infectious virus, used for the studies reported below.
Mice
C57BL/6 mice (H-2b MHC haplotype) were acquired from The Scripps Research Institute (TSRI) breeding facility, and µMt/µMt (-/-) mice were purchased from Jackson Laboratories and bred at TSRI. The µMt mice (also H-2b) are B-cell-knockout (BcKO); a membrane exon of the gene encoding the µ chain was deleted, and B cell development in the resulting homozygous mice is arrested at or before the pre-B cell stage.12 All mice used were 8 weeks of age or older.
Organ Preparation for Titrations and Histology
Immediately before removal of organs, mice were injected with 500 µl of 3.5% chloral hydrate. Mice were then perfused with normal saline to clear blood from organs, thus preventing overestimation of organ viral titers resulting from blood-borne CVB3. Heart, pancreas, liver, kidney, lung, spleen, and brain were removed from mice; half of each organ was placed in a cryotube and fast-frozen in liquid nitrogen for titrations. These materials were weighed and subsequently homogenized in 1 ml of serum-free Dulbecco's modified Eagle's medium (DMEM). The other half of the organ was fixed (Bouin's or 10% normal buffered formalin) and embedded in paraffin for sectioning before appropriate histological analysis. Immunohistochemistry was carried out using 6-µm frozen sections.
Plaque Assays
Six-well plates were plated with 7.5 x 105 HeLa cells/well 24 hours before infection and grown overnight in a 37°C incubator with 5% CO2. Cells were 90 to 100% confluent at the time of titration. Organs to be titered were serially diluted (10-fold) in serum-free DMEM. Media was aspirated, and 400 µl of serially diluted homogenized organ was added to each well. Infected plates were placed back in the incubator for 1 hour and rocked every 10 minutes. After 1 hour, the inoculum was removed by aspiration, and cells were overlaid with 3 ml of 1x DMEM in 0.5% agar (1:1 mixture of 2x DMEM (Gibco-BRL, Gaithersburg, MD) at 37°C and 1% agar at 55°C). Between 40 and 50 hours after infection, cells were fixed by adding 2 ml of methanol:acetic acid (3:1 v/v) to each well and letting the plate sit for 10 minutes. The fixative was poured off, and agarose plugs were removed. Cells were stained with 1 ml 0.5% crystal violet in 20% ethanol per well. Plates were rinsed in tap water and plaques were counted.
Cell Sorting before Infectious Center Assay
Splenocytes from infected C57BL/6 mice were harvested 1, 2, 3, or 4 days after infection, and a single-cell suspension of splenocytes was prepared by homogenization, followed by lysis of red blood cells (5 minutes at room temperature, 0.83% NH4Cl). Polystyrene petri dishes (100 x 15mm) were coated with anti-Ig antibodies by incubation with 5 ml of buffer (0.05 mol/L Tris-HCl, 0.15 mol/L NaCl, pH 9.5) containing 16 µg of anti-Ig antibody and 350 µg of bovine serum albumin at room temperature for 1 hour or longer, followed by washes with phosphate-buffered saline (PBS) and PBS + 5% serum. Approximately 3 x 107 splenocytes, resuspended in 3 ml of PBS + 5% serum, were incubated on the coated dishes for 1 hour at 4°C. The B-cell-depleted supernatant was incubated on a second plate with 4 times more antibody to ensure complete depletion of B cells. The cells attached to the dish were washed with ice-cold PBS + 5% serum and then recovered in 5 ml of PBS + 5% serum (37°C) by vigorous pipetting. The efficiency of the depletion/enrichment was assessed by staining with fluorescein isothiocyanate (FITC)-labeled anti-B220 antibody followed by fluorescence-activated cell sorter (FACS) analysis.
Infectious Center Assay
Total splenocytes, as well as the B-cell-enriched and the B-cell-depleted fractions, were counted using Trypan blue, and serial 10-fold dilutions were made. One hundred microliters of each dilution were added to HeLa monolayers, allowed to settle, and overlaid with agar-containing medium. After 36 hours' incubation, cells were fixed and stained and plaques were counted. Each plaque indicates a single productively infected cell.
Preparation of RNA and Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
Total liver RNA was obtained using TRIZOL reagent (Gibco-BRL) and the conditions recommended by the manufacturer. Purified B cell mRNA was obtained as follows: 5 x 106 splenocytes were mixed with 1.2 x 108 magnetic beads (PerSeptive Biosystems, Framingham, MA) coupled to anti-B220 antibody. After incubation, B220+ cells were selected using a magnetic field (MPC-2 magnet, Dynal, Lake Success, NY). Poly(A)+ RNA from the enriched B cells was obtained using Dynal's mRNA Direct Kit, and the conditions recommended by the supplier. Briefly, B cells were lysed, and the lysate was incubated with oligo(dT)-coupled magnetic beads. The Dynal MPC-2 magnet was used to select the poly(A)+ RNA which was washed and eluted in low salt buffer at 65°C, and used as template for the synthesis of first strand cDNA using oligo(dT) as primer and RT Superscript II (Gibco). The first strand cDNA was used in a PCR reaction (35 cycles) with primers specific for the mCAR gene (mCAR1, 5'-GGCGCGCCTACTGTGCTTCG-3'; mCAR2, 5'-CTGCCAGCCATGGCGTAGGC-3') or for the housekeeping gene glyceraldehyde phosphate dehydrogenase (GAPDH1, 5'-CCATCACCATCTTCCAGGAG-3'; GAPDH2, 5'-CCTGCTTCACCACCTTCTTG-3').
In Situ Hybridization (ISH)
CVB3-specific, P33-labeled, RNA probes were transcribed in vitro from a linearized plasmid containing a 421-base fragment from the capsid region of CVB using RNA polymerase T7 to generate negative sense probe or T3 to generate positive sense probe. A previously published protocol13 was followed using 5-µm paraffin sections from tissues previously fixed in 10% neutral buffered formalin. After preparation of the slides and prehybridization at 42°C, 2 x 106 to 5 x 106 cpm of the P33-labeled RNA probe was applied and allowed to hybridize overnight at 45°C. After washing, slides were dipped in photographic emulsion and held at 4°C for 1 to 7 days, when they were developed and fixed. Slides were then stained with hematoxylin (3–5 minutes) and eosin (2–3 minutes) and mounted.
Adoptive Transfers
C57BL/6 mice, previously infected with 500 plaque forming units (pfu) of CVB3, were killed at >4 weeks after infection and their spleens were removed and placed immediately in complete 1x DMEM (DMEM supplemented with 10% fetal bovine serum, 2 mmol/L L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin). Spleens were disrupted into a single-cell suspension and red blood cells were lysed using 0.83% ammonium chloride lysing buffer. Pan-B cells and pan-T cells were isolated from splenocytes using VarioMACS magnetic cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Samples of both B cell and T cell populations were FITC-labeled for FACS analysis to evaluate the efficiency of sorting. Recipient mice were uninfected BcKO mice that received one of six sets of inocula by the i.p. route: (i) no cells, (ii) B cells from an immune mouse, (iii) T cells from an immune mouse, (iv) a combination of B cells and T cells from an immune mouse, (v) B cells from a non-immune mouse, or (vi) T cells from a non-immune mouse. In all cases where cells were given, each recipient mouse received 0.5 x 107 to 4 x 107 lymphocytes (precise numbers varied between experiments, but all gave similar results). Immediately after adoptive transfer injections, all mice were challenged with 5000 pfu CVB3 i.p. All mice were monitored for survival, and serum CVB3 titers were done at days 4, 7, 14, 21, and 28. In a separate experiment, two chronically infected BcKO females were identified by the presence of high titers of virus at 21 days after infection; at 26 days postinfection both females were injected i.p. with 5.4 x 107 B cells from an immune C57BL/6 mouse. Virus titers were measured at the indicated times, and both mice were sacrificed at day 21 posttransfer for organ viral titers and histology.
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Others have reported that, in immunocompetent mice, CVB can be
detected in splenic lymphocytes, particularly B cells, during the acute
stage of infection.7,8
However, the mere identification of
virus RNA or protein within cells is not proof of productive infection
because, at least in tissue culture, CVB can establish persistent
infection in several cell types,14,15
including lymphoid
cells.16
Although one study showed that splenocytes were
productively infected,7
the precise source of nascent
virus was not identified, because cell sorting was not carried out
before assay, nor were individual cells evaluated for virus production.
To determine whether B cells were productively infected by CVB, we
sorted the splenocytes from infected C57BL/6 mice at 1, 2, 3, and 4
days after infection (4 mice per time point), separating them into B
cell and non-B cell populations, which were then evaluated by
infectious center assay. For each cell population, dilutions of cells
were plated on a HeLa cell monolayer, then immobilized by an agarose
overlay. The development of plaques was observed by microscopy, the
monolayers were fixed and stained, and the plaques were counted. As
shown in Figure 1
, no infected cells were
detected on day 1, but on days 2 and 3, approximately 1% of both cell
populations scored positive for productive CVB infection. By day 4
after infection the number had decreased slightly, to approximately
0.5%. These data show that B cells are indeed productively infected;
however, the frequency of B cell infection appears very similar to that
of non-B cells. Note that positive controls were carried out in
parallel to determine the sensitivity of the infectious center assay.
HeLa cells were infected with a known number of pfu, and these infected
cells were diluted and plated on an uninfected HeLa monolayer. However,
the number of cells scoring positive usually were ~10% of the number
expected from the input pfu. Therefore, the absolute values in these
experiments may be ~10-fold underestimates of the percentages of B
and non-B cells productively infected.
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The productive infection of immune cells by CVB3 might have
serious biological consequences for the host. Perhaps the most obvious
is immunosuppression; transient global immunosuppression has been
reported in this infection,9
and it was suggested that
this resulted from defects in splenic function, perhaps antigen
presentation.10
In these early studies the infection of
lymphocytes, although sought, went undetected. Our studies, together
with those of other labs,7,8
suggest that at any given
time in the acute infection, 1 to 10% of B cells are productively
infected by this lytic virus. To exclude the possibility that
large-scale depletion of B cells was effected by the virus, we carried
out FACS analyses of the spleen at up to day 21 after infection; as
shown in Figure 2
, the proportion of B
cells increased transiently at 3 days after infection, most likely the
result of virus-specific B cell proliferation in germinal centers, but
remained quite stable thereafter, demonstrating that acute CVB
infection does not dramatically reduce splenic B cell levels.
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Thus, it appears that B cells are productively infected early in
CVB infection, but that their numbers remain relatively stable as
infection progresses. Do these cells act as an important primary target
for CVB replication and/or might they be implicated in virus
dissemination to other host tissues? To investigate this, we compared
C57BL/6 mice with BcKO mice, which lack mature B lymphocytes. C57BL/6
or BcKO mice were infected, and CVB titers were measured at 12 hours
and 1, 2, and 3 days postinfection. As shown in Figure 3
, virus replication/dissemination
appeared delayed in BcKO mice. By 24 hours after infection a difference
was readily detected; for example, virus was presented in the hearts of
C57BL/6 mice, but not of BcKO mice. Of 10 tissue samples analyzed in
each mouse strain at this time point, only 3 were positive in BcKO
compared to 9 in C57BL/6; titers were much higher in the latter,
reaching a maximum of ~4 x 109
pfu/gram
(pancreas) compared to a maximum almost 50,000-fold lower in the
pancreas of one BcKO mouse. Marked differences were also noted in the
serum and spleen. At day 2 after infection virus titers in serum,
heart, and spleen of BcKO mice were 10-fold to 1000-fold lower than in
the same tissues from C57BL/6 mice. By 3 days after infection, viral
titers in BcKO mice were similar to those in immunocompetent mice,
suggesting that viral replication cannot be constrained in the absence
of antibody. These data show definitively that mature B cells are not
required for CVB3 infection, but suggest strongly that these cells play
a role early in infection, in providing the virus with a susceptible
cell population and/or in disseminating the virus to distant tissues
such as heart, spleen, and pancreas. Similar results were seen at early
times in a separate experiment (see Figure 6
).
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If B cells are an important focus of primary infection,
one might anticipate differences in the distribution of CVB3 in the
spleen when comparing BcKO mice with their immunocompetent
counterparts. We used ISH to compare virus-infected cells in the
spleens of immunocompetent and BcKO mice at days 2 and 3 postinfection.
As shown in Figure 4, A and D
, virus was
difficult to detect by ISH in either mouse strain at 2 days after
infection, although in C57BL/6 mice, which had a splenic titer of
~5 x 108
pfu/gram (Figure 3)
, a weak
signal was detected in the marginal zone surrounding the white pulp
(Figure 4B)
. In contrast, the next day virus was readily detectable in
the spleen of C57BL/6 mice (Figure 4E)
, mainly in the outer areas of
the white pulp (Figure 4F)
, an area comprising predominantly B cells
(Figure 4G)
. Despite the much stronger ISH signal on day 3 after
infection, the splenic virus titers in C57BL/6 mice are similar on both
days, suggesting altered distribution of the infectious materials in
the spleen; this has previously been described.7,17
The
strong day 3 signal was absent from the spleen of BcKO mice (Figure 4H)
, consistent with the signal being predominantly in B cells. At this
point it is impossible to determine whether the infected B cells seen
in C57BL/6 mice in Figure 4
were infected in the spleen or whether they
were infected in an extra-splenic area and later congregated in the
spleen.
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To continue our investigation of CVB infection of splenic cells,
we sought evidence of CVB receptor expression on the surfaces of B
cells. Many enteroviruses,18
including
coxsackieviruses,19,20
bind to the cell surface molecule
decay accelerating factor (DAF), but DAF binding is insufficient for
internalization; the latter requires another
protein,21
also important for internalization of
adenovirus, and thus termed the coxsackievirus-adenovirus receptor
(CAR).22,23
The murine homologue, mCAR, has recently been
cloned,24
and the gene was generously provided to us by
Dr. J. Bergelson. An RNA probe complementary to the ORF was used to
evaluate mCAR mRNA levels in the spleens of C57BL/6 and BcKO mice by
ISH, but no convincing signal was seen (not shown), leading us to
attempt to identify mRNA expression in purified B cells using RT-PCR.
As a positive control template we used total RNA from liver, a tissue
known to be strongly positive for mCAR.24
After 35 PCR
cycles, mCAR mRNA signal from total liver RNA was easily visualized on
an ethidium-stained gel (Figure 5A)
and
was overwhelmingly positive on Southern blot (Figure 5B)
. In contrast,
despite using oligo-dT-selected mRNA as template, the mCAR signal from
purified B cells was invisible on the ethidium-stained gel (Figure 5A)
and only very weakly positive on probing of the Southern blot (Figure 5B)
. The quality of the oligo-dT-selected B cell mRNA was confirmed
using control primers for the mRNA encoding the housekeeping enzyme
GAPDH (Figure 5A)
. Given the extraordinary sensitivity of RT-PCR
followed by Southern blotting, it is difficult to conclude with
certainty that this weak mCAR signal is B-cell-derived; it is possible
that the signal comes from a low level of contamination by non-B cells.
Identical RT-PCR analysis of total splenocytes also gave only a weak
signal (not shown).
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The preceding studies compare immunocompetent and BcKO mice during
the acute phase of infection and show differences in early virus titers
(Figure 3)
. This result is confirmed in Figure 6
, which also demonstrates the profound
differences seen later in infection in BcKO mice. By day 14 after
infection CVB3 had, as expected, been cleared from most organs in the
C57BL/6 mice, whereas viral titers were high in BcKO mice. At 45 days
after infection virus was undetectable in C57BL/6 mice but remained at
high titers in all BcKO organs titered: heart, pancreas, brain, liver,
kidney, lung, spleen, and serum. The chronically infected BcKO mice
exhibited visible steatorrhea and profound wasting (perhaps the result
of the malabsorption) and rarely survived beyond 60 days.
Severe Myocardial Damage in Chronically Infected BcKO Mice
The capacity of CVB to cause acute myocarditis and subsequent
myocardial fibrosis in mice is well recognized; we have shown that both
processes are exacerbated by CD8+ T
cells25
and, more specifically, require
perforin.26
BcKO mice can mount apparently normal
CD8+ T cell responses27
and one
might expect that these mice would develop myocardial lesions. This is
indeed the case. Acute myocarditis develops by 7 days after infection
(not shown) and is maintained as ongoing myocarditis with concurrent
fibrosis. By 45 days after infection severe scarring is visible, along
with ventricular dilation (Figure 7A)
.
The fibrosis extends widely throughout the myocardium, and in some
places spans at least 50% of the ventricular wall (Figure 7B)
. ISH
using a negative sense (genome-detecting) probe shows a focal pattern
of virus distribution (Figure 7, C and D)
similar to that seen in the
acute phase in immunocompetent mice.25
Despite the high
virus titers and easily detected genomic-sense viral RNA, use of a
positive sense probe yields a barely detectable signal (Figure 7, E and F)
, indicating that there is a great excess of genome-sense RNA
compared to antigenomic material. Others have shown that, during acute
CVB infection when high levels of infectious virus are produced, the
ratio of CVB genome to antigenome is approximately 100:1, but when
persistence is established in immunocompetent mice, infectious virus is
undetectable and the genome:antigenome ratio drops to around
1:1.28
The virus RNA present in BcKO mice at 45 days after
infection is predominantly genomic (compare Figure 7, C and D
to Figure 7, E and F
), suggesting that the virus replication strategy remains as
in acute infection, consistent with the high titers of virus shed.
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Uninfected BcKO mice received one of six sets of cells by adoptive
transfer; no cells, B cells, or B and T cells from non-immune C57BL/6
mice; or B cells, T cells, or B and T cells from CVB-immune C57BL/6
mice. The following day the BcKO mice were challenged with CVB and
virus titers in serum were evaluated. As shown in Figure 8A
, all mice developed viremia by 4 days
after infection, and several died within a few days of infection. Of
the surviving mice, none of those which received cells from a
non-immune host cleared the virus, while 8 of 13 mice primed with
immune cells showed transient (5 mice) or permanent (3 mice) clearance
of detectable virus in the serum. Of these 8 mice, 7 received immune B
cells (5 received B and T cells, 2 received only B cells); the eighth
mouse received T cells alone. That B cells appear more effective than T
cells in this assay is reflected by the fact that 100% (7 of 7 mice)
of B cell recipients surviving beyond day 6 cleared virus at least
transiently, whereas this occurred in only 55% of surviving T cell
recipients (6 of 11 mice; 5 of these 6 mice had also received B cells).
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The above demonstrates that immune cells can limit acute viral
replication in BcKO mice, sometimes effecting complete viral clearance.
To confirm the therapeutic benefit of immune B cells and to extend the
finding into a different clinical situation, 2 BcKO mice chronically
infected with CVB3 were adoptively transferred with immune B-cells. We
have shown previously that such chronically infected mice do not
spontaneously clear CVB. As shown in Figure 8B
, within 4 days of B cell
transfer, virus was undetectable in the serum of both mice, and this
clearance was accompanied by an improvement in the appearance of the
mice (increased locomotor activity, normal posture, no ruffled fur).
The serum clearance was maintained for at least 21 days, when the mice
were killed and their organs titered. No virus was detected in any of
the titered tissues (liver, heart, lung, spleen, brain, pancreas,
kidney).
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. Furthermore,
virus appears to replicate in splenic B cells. B cells enter the spleen
through the splenic artery and migrate through the marginal zone into
the white pulp; CVB3 infection is first detected in the spleen at 2
days after infection, around the edge of the white pulp, in the
marginal zone (Figure 4B)
, and it appears likely that replication
within these cells leads to the stronger splenic ISH signal at day 3
after infection, seen only in B-cell-containing mice (Figure 4, E and F)
. The change in location of the ISH signal between day 2 and day 3 is
consistent with migration of the infected B cells into the white pulp
over this 24-hour period. In addition to being an early site of
infection and replication, B cells play a role in virus dissemination,
as demonstrated by the delayed spread at early times in BcKO mice
(Figures 3 and 6)
. Particularly striking is the finding that virus
titers in solid tissues (eg, heart and pancreas) at 1 day postinfection
are 103-fold to 105-fold
lower in mice deficient in B cells. The potential benefits to the virus
of replicating inside mobile host cells such as lymphocytes are clear;
it can replicate while in transit to distant tissues, and at the same
time sequester itself from neutralization by antibodies. This "Trojan
horse" hypothesis is consistent with the recent observation that B
cells containing CVB RNA are detectable within the
myocardium.29
However, although B cells play host to CVB3
in normal mice, virus dissemination does not require their presence, as
shown by our demonstration of successful infection by and dissemination
of CVB in mice lacking B lymphocytes (Figures 3 and 6)
. Presumably, any
role of B cells in dissemination can be paralleled by other
splenocytes, albeit with a slight delay. Our infectious center analyses
show that, at several time points after infection, the frequencies of
productive infection in non-B cells are comparable to those of B cells
(Figure 1)
; these data are underlined by the observation that splenic
titers are similar at days 2 and 3 in C57BL/6 mice, despite the marked
difference in ISH B cell signal. Thus, we speculate that the major
benefit to the virus of B cell infection might lie not in virus
dissemination, but instead in evasion of the host response. All
mammalian viruses so far studied attempt to escape the host immune
response, in many cases by disarming the aspect of the immune response
that most strongly opposes them. For example, antibodies play a
critical role in immunity to influenza viruses, and in response to this
host pressure, the viruses undergo antigenic drift and shift.
Conversely, herpesviruses, adenoviruses, and lymphocytic
choriomeningitis virus are controlled mainly by T cells, and these
viruses make strenuous attempts to avoid the unwelcome attention of T
lymphocytes. As described above, antibodies play a vital role in
controlling enteroviral infections, and the strikingly high titers of
CVB3, which are maintained for up to 60 days in BcKO mice, underline
the critical role played by B cells in controlling this infection. It
is therefore tempting to suggest that the infection by CVB of B cells
(and, perhaps, of other immunologically important cells) represents an
attempt by the virus to undercut this antagonistic immune response.
Studies published more than two decades ago suggested that
coxsackievirus infection results in suppression of humoral immune
responses.9
We show here that CVB infection does not
effect a wholesale depletion of B cells, but other possible
immunosuppressive consequences of CVB infection of immune cells are
under investigation. It will be interesting to determine the antigen
specificity of the B cells that become infected; it has been shown in
the lymphocytic choriomeningitis virus (LCMV) model that B cells
specific for the viral glycoprotein are specifically deleted,
apparently because B cells displaying glycoprotein -specific antibodies
are selectively infected, and subsequently destroyed by anti-LCMV
cytotoxic T lymphocytes.30
For many years it was considered likely that, for any given virus, a
single cell surface protein would act as its receptor. However, it is
now recognized that, for several viruses, cell binding and entry may
require more than one protein, acting serially or in concert. Many
enteroviruses,21,31
including CVB types 1, 3, and
5,20
bind to the cell surface protein CD55 (DAF, a member
of the complement regulation system). However, DAF binding does not
invariably result in infection20
and, conversely, some
CVB3 isolates fail to bind DAF, but nevertheless can infect and cause
disease in mice.19
Recently a second protein has been
identified that appears to be critical to cell entry.22,24
This protein, also used by adenovirus and hence termed the
coxsackievirus-adenovirus receptor (CAR), is widely distributed (as
judged by Northern blot analysis of RNA expression), having been found
on liver, heart, lung, and kidney; however, it is barely detectable
in human spleen, and was not detected in mouse
spleen.24,32
Because we found extensive productive B cell
infection, we evaluated mCAR mRNA expression in B cells and found it to
be extremely low, detectable only after Southern blot analysis of PCR
products (Figure 5B)
. These results suggest that mCAR may not be
required for B cell infection. Because the tissue tropisms of
coxsackievirus and adenovirus are markedly different, it seems likely
that additional factors, rather than CAR, are the ultimate determinants
of coxsackievirus and adenovirus tropism. How else might CVB enter B
cells? DAF is one candidate and is expressed on B cells. A recent study
implicated complement component 3 in localization of CVB3 antigen to
splenic germinal centers, and the authors suggested that this
component may act as a bridge through which DAF binds
CVB.17
Our findings are consistent with this hypothesis
and suggest that such interactions may permit B cell infection,
obviating the need for mCAR.
CVB infection in mouse and man can result in both acute and chronic
myocarditis, the latter sometimes leading to dilated cardiomyopathy. In
chronically infected BcKO mice the heart was severely scarred and
dilated (Figure 7)
, as is often seen in human dilated cardiomyopathy,
which is one of the most serious sequelae of acute viral myocarditis
and which can be treated effectively only by heart
transplantation.33,34
The mechanism underlying the human
chronic inflammatory disease is controversial. Although most observers
agree that there is a large immunopathological component, some groups
believe it to be autoimmune; others advance virus-specific
explanations. CD8+ T cells greatly exacerbate
acute myocarditis25
by perforin-mediated lysis of infected
cells.26
We are currently investigating whether the
chronic myocarditis seen in BcKO mice is dependent on
CD8+ T cells. Others have suggested that
CVB-induced myocarditis may be mediated by anti-myosin antibodies
induced by CVB infection,35
although this is
controversial.36
The data in the present study demonstrate
clearly that the ongoing myocarditis in BcKO mice cannot be
antibody-mediated. If the chronic myocarditis is indeed driven by
virus-specific immune responses, rather than by autoimmune phenomena,
then at the very least some CVB antigen must be present in the target
cells. Although CVB infection, in common with most picornaviruses, is
often rapidly cytolytic, there is solid evidence that the virus can
persist in tissue culture cell populations continually passaged for 1
year.15,16
Here we show that long-term virus shedding
takes place in B-cell-deficient mice. Others have previously
demonstrated long term infection in T-cell-deficient
mice.37,38
Thus, chronic CVB infection can occur in
vivo. However, is this limited to immunodeficient mice? Although
there appears to be little doubt that viral RNA can be detected up to
at least 30 days postinfection in several immunocompetent mouse
strains,28,39
there is little evidence in such mice of
long-term productive infection. We have confirmed the presence of CVB
RNA in various organs of C57BL/6 mice up to 45 days after infection,
but we have not succeeded in isolating infectious virus (Mena, Fischer,
and Whitton, unpublished). However, other picornaviruses (for
example, Theiler's virus), can persist for many months in
immunocompetent mice, and it would be premature to exclude this
possibility for coxsackieviruses. In humans there is little evidence
that infectious virus persists, and although enteroviral RNA sequences
have been reported in various tissues long after the apparent primary
infection,40
even this issue is
controversial.41
But many studies of human heart used
myocardial biopsy material which is, for obvious reasons, limited in
quantity. As we show in Figure 7
, CVB distribution is very focal even
at a titer of 108
pfu/gram of heart tissue, so
perhaps it is not surprising that CVB materials sometimes cannot be
identified in biopsies of hearts in which infectious virus is
undetectable.
Several of our findings in BcKO mice parallel those in human X-linked
agammaglobulinemia, and thus may serve as a model with which to
evaluate novel approaches to the management of this syndrome, in which
susceptibility to microbial infection is a major concern. Many of these
infections are established by pyogenic bacteria, but certain virus
infections also are more common. For example, agammaglobulinemics are
susceptible to chronic enteroviral infections3,4
; they may
continue to shed virulent poliovirus for many years following live
virus vaccination42
; and CVB can establish long-term
productive infection with encephalitis.5,6
In the present
study we demonstrate long-term productive CVB infection of several
tissues including the brain (Figure 6)
. We have previously shown that
CD8+ T cells reduce CVB load by up to
95%,25
and BcKO appear to mount normal
CD8+ T cell responses,27
although
they may have ancillary immune defects.43
However, the
chronic infection of CVB3 in BcKO mice shows that
CD8+ T cells alone cannot control CVB infection
in the absence of antibodies, underlining the enormous importance of
humoral immunity in the control and eradication of this virus. During
acute CVB infection the ratio of CVB genome to antigenome is
approximately 100:1. Studies have suggested that, when persistence is
established in immunocompetent mice, the genome:antigenome ratio
changes to around 1:1.28
The virus RNA present in BcKO
mice at 45 days after infection is predominantly genomic (compare
Figure 7, C and D
to Figure 7, E and F
), consistent with the high
titers of virus shed. Thus, in BcKO mice the virus maintains an acute
replication scheme. We suggest that, in normal mice, the antibody
response imposes on the virus the requirement that it alter its
replication strategy, resulting in a 1:1 genome:antigenome ratio and
undetectable infectious virus, whereas in BcKO mice (or in
agammaglobulinemic humans), the lack of selective pressure from
antibodies allows the virus to retain a 100:1 genomic ratio, along with
the high levels of infectious virus. The BcKO mouse therefore appears
to be a good model for enterovirus infection in human
agammaglobulinemia. Further adoptive transfer studies may help us
identify the mechanisms underpinning CVB persistence in an
immunocompetent host.
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Acknowledgements |
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Footnotes |
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Supported by National Institutes of Health grant AI-42314.
Accepted for publication June 3, 1999.
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References |
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