Institute for Animal Health, Compton, Newbury RG20 7NN, UK1
Author for correspondence: Bryan Charleston. Fax +44 1635 577263. e-mail bryan.charleston{at}bbsrc.ac.uk
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Previous studies of animals infected with pestiviruses have highlighted the presence of the viral genome and/or antigen in the germinal centres of lymphoid tissue (Bielefeldt Ohmann, 1983 , 1988
; Collins et al., 1999
; Desport et al., 1994
; Susa et al., 1992
). The classical swine fever virus (CSFV) genome has been localized to germinal centres after acute infection and, on the basis of this observation, it has been suggested that germinal centres are the site of CSFV replication (Susa et al., 1992
). Sections of lymph nodes from cattle clinically affected by mucosal disease, probed with anti-BVDV polyclonal sera, showed heavy staining of germinal centres, particularly associated with the long cytoplasmic processes of large cells considered to be follicular dendritic cells (FDC) (Bielefeldt Ohmann, 1983
). More recently, the BVDV genome and the glycoprotein Erns have been localized to germinal centres in PI animals (Desport et al., 1994
; Collins et al., 1999
). More detailed aspects of the latter study (Collins et al., 1999
) focussed on PI animals with detectable circulating anti-BVDV antibodies. In the present study, we set out to define the precise location of BVDV antigen and genome in the germinal centres of experimentally produced PI calves that did not have detectable circulating antibody to BVDV.
BVDV antibody-positive cows were presented for in utero infection at approximately 60 days of pregnancy (range 5870 days): 5x106 TCID50 of ncp BVDV (Pe515ncp; Brownlie et al., 1984 ) was injected directly into the amniotic fluid. The cows were maintained in isolation from other cattle for the remainder of pregnancy. Calves were derived by hysterotomy at approximately 10 days before term into disease-secure accommodation, which ensured that there was no risk of extraneous infection. Each calf was confirmed to be persistently viraemic with BVDV and seronegative to BVDV (Brownlie et al., 1984
). Prescapular lymph nodes were surgically removed at either 1 day of age (n=2) or approximately 2 months of age (n=4). Lymph nodes were also sampled from age-matched animals that had been shown to be negative for BVDV and antibody. Samples of each lymph node were either preserved in 4% paraformaldehyde for histological sectioning or mounted in OCT compound (Miles), frozen and stored at -70 °C for cryosectioning. In situ hybridization of sections prepared from wax-embedded tissues was performed as described previously (Lennard et al., 1995
), using an 35S-UTP-labelled riboprobe transcribed from a 1·1 kb fragment of E2 cDNA from Pe515ncp, cloned in pTarget (Promega). The distribution of BVDV antigens was examined by indirect immunofluoresence (IIF) (Fray et al.,1998
) using three anti-BVDV MAbs, WB103, WB162 and WB210, which are specific for the non-structural protein NS3 and the envelope glycoproteins E2 and Erns, respectively (Paton et al., 1997
). Cryostat sections of prescapular lymph nodes from BVDV-positive and BVDV-free calves were stained with the MAbs and a control MAb of the same isotype (IgG1) raised against turkey rhinotracheitis virus-1, followed by FITC-labelled goat anti-mouse IgG. The control antibody did not give any staining in tissues from infected or uninfected animals and no staining was observed with the BVDV-specific antibodies in tissues from uninfected animals.
Histological comparison of lymph nodes from PI and uninfected calves did not reveal any gross differences in the structure of the follicles and germinal centres present. In sections of lymph node from the PI animals, individual cells staining for E2 and NS3 (Fig. 1e, g
) were observed scattered throughout the lymph node, including the B cell follicles, paracortex and medullary regions. There was also a similar distribution of cells that stained weakly for Erns in addition to more intense and diffuse staining for Erns within the germinal centres of the B cell follicles (Fig. 1c
). This latter staining was often focussed in the anterior pole of the germinal centre, proximal to the lymph node capsule. In situ hybridization showed that distribution of the BVDV genome was similar to that of Erns with intense staining in the germinal centres of the B cell follicles (Fig. 1a
, b
and c
) as well as more widespread but weaker staining of individual cells. Compared to animals housed in a conventional environment, the lymph nodes from animals in this study contained relatively few germinal centres. The lymph nodes from both the PI and uninfected neonatal calves had very few primary and secondary B cell follicles. However, a large proportion of the secondary follicles from the PI animals exhibited diffuse staining for BVDV RNA and Erns antigen in the light zone of their germinal centres. The lymph nodes from the two older calves examined in this study had a greater number of secondary B cell follicles than the neonatal calves, but only a minority of the germinal centres were positive for BVDV RNA and Erns antigen. A positive-sense version of the E2 riboprobe did not hybridize to sections from viraemic animals. Sections from uninfected animals did not stain with either positive- or negative-sense riboprobes.
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Lymph node sections from viraemic animals stained for Erns with MAb WB210 and anti--tubulin MAb (clone B-5-1-2; Sigma). The majority of the staining for Erns did not co-localize with
-tubulin (Fig. 1d
). This failure to co-localize Erns with
-tubulin is in contrast with the almost complete co-localization of NS3 and E2 with
-tubulin (Fig. 1f
, h
).
Sera collected from the six PI calves from which lymph nodes were examined were negative for anti-BVDV antibody as determined by virus neutralization (Brownlie et al., 1984 ) and ELISA (Howard et al., 1985
). In an attempt to detect low levels of antibody in the form of immune complexes in the serum, 7% PEG was added to the serum from one of the calves to precipitate immune complexes (Azazy et al., 1994
). Immunoglobulin was purified from the re-dissolved PEG precipitates using a protein G column (Pharmacia). Elution of antibodies from the column and disruption of immune complexes were performed simultaneously using 0·1 M glycine, pH 2·0. The column eluate was immediately neutralized using one tenth volume 1 M Tris, pH 7. The eluted material was examined for the presence of BVDV-specific antibodies by immunoprecipitation of [35S]methionine-labelled proteins from cells infected with cp or ncp Pe515 BVDV, or mock-infected cells (Harlow & Lane, 1988
). Sera from a BVDV-hyperimmune animal and an animal that had not been exposed to BVDV were used as control antibodies in the immunoprecipitation assays. No evidence of anti-BVDV antibody was found in the immunoglobulin obtained from PEG precipitates (Fig. 3
). However, this does not rule out the possibility that small amounts of antibody of low avidity are being produced.
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An unexpected finding was the absence of detectable E2 protein in the germinal centre deposits. We also failed to detect E2 in the germinal centre using MAbs that recognize a number of different epitopes on the E2 molecule. Similar findings have been reported by Collins et al. (1999) . Antigen localized on the surface of FDC in the form of immune complexes is usually preserved for months in its native conformation. The trapped antigen acts as a long-term reservoir for the induction and maintenance of B cell memory responses (Tew et al., 1997
; Bachmann et al., 1996
). The possibility that E2 epitopes may be masked by an excess of antibody within the germinal centres is unlikely, given the absence of detectable circulating BVDV-specific antibodies in these animals. A recent study by Weiland et al. (1999)
reported that E2 epitopes on BVDV viral particles could not be detected by immunoelectron microscopy when the virions were associated with the cell membrane. Hence, the absence of detectable E2 in germinal centres may be due to conformational alteration upon association with cells in the germinal centres.
Persistent infection with BVDV is established when a calf is infected in utero prior to the onset of immune competence. The absence of circulating antibody and the sustained viraemia in PI calves has been taken as evidence that such animals are immunologically tolerant to the virus. We have also found that PI calves do not give detectable in vitro T cell proliferative responses to the persistent virus (T. Collen and I. Morrison, unpublished data). However, the detection of viral antigen co-localized with immunoglobulin in the majority of the germinal centres in lymph nodes of PI neonatal calves implies that there is a virus-specific B cell response, although careful analysis of serum from the animals failed to reveal any specific circulating antibody. This is in contrast to the study by Collins et al. (1999) , who reported the detection, in a proportion of PI animals, of antibody that neutralized the persisting ncp virus. These authors also found evidence of variation in the RNA sequence in two of the animals with circulating antibody, and proposed that changes in B cell epitopes as a result of mutation of the virus were responsible for inducing the antibody response. However, this conclusion is at odds with their observation that serum antibody neutralized the persisting virus, within which the mutant viruses could be expected to represent only a minor component.
The development of fully differentiated germinal centres following trapping of immune complexes by FDC is dependent on specific T cell responses (Liu & Arpin, 1997 ). The germinal centres identified as containing viral antigen and RNA, in our studies, were not obviously different from germinal centres in normal antigenically stimulated lymphoid tissues, in that they were of similar size, had clearly differentiated light and dark zones and contained small numbers of CD4+ cells. The implication of these findings is that both B and T lymphocytes reactive with the virus are involved. The formation of germinal centres might, therefore, be a consequence of the incomplete immunological tolerance to the virus or result from spontaneous generation of mutations in the virus that give rise to T cell epitopes capable of providing help for differentiation of virus-specific B cells. The latter model assumes that B cell tolerance is incomplete. Incomplete B cell tolerance has been demonstrated in mice infected, either in utero or neonatally, with lymphocytic choriomeningitis virus (Oldstone & Dixon, 1967
). These mice had persistently high titres of virus in their blood and no detectable circulating antibody. However, immune complexes could be detected in the kidneys. The absence of detectable BVDV-specific antibody in the serum may be due to consumption of the antibody by an excess of viral antigen in the circulation, although we have failed to detect immune complexes in serum or in the kidneys (data not shown). Alternatively, it is known that B cells in the germinal centres undergo apoptosis and/or are excluded from the germinal centres if exposed to large quantities of soluble viral protein (reviewed by Bachmann, 1998
). In this way, B cells may be eliminated before they expand to the level where sufficient detectable immunoglobulin is produced in the circulation. The latter situation is potentially similar to that seen in calves persistently infected with BVDV, in which large quantities of viral antigen are present in the blood. We therefore conclude that immune complexes containing Erns are present in specific germinal centres of PI animals. We hypothesize that the presence of these immune complexes is due to incomplete B cell tolerance, and that the failure to detect circulating antibodies is the result of failure in maturation of the B cell response coupled with the high load of soluble antigen in these PI animals. More detailed studies of the antigenic specificities of B cells in the lymphoid tissues are required to address this hypothesis.
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Received 29 October 1999;
accepted 21 March 2000.