1 Department of Biotechnology, National Institute for Agriculture and Food Research and Technology, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Ctra Coruña km 7·5, 28040 Madrid, Spain
2 Virus Receptor and Immune Evasion Group, Department of Medical Biochemistry and Immunology, Cardiff University School of Medicine, Third Floor Henry Wellcome Research Institute, Heath Park, Cardiff CF14 4XN, UK
Correspondence
O. Brad Spiller
SpillerB{at}cardiff.ac.uk
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ABSTRACT |
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Present address: Departamento de Enfermedades Emergentes, Laboratorio Central de Veterinaria, Ctra Algete km 8, 28110 Algete (Madrid), Spain.
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INTRODUCTION |
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SVDV is a porcine variant of the human pathogen coxsackie B virus serotype 5 (CVB5) (Brown et al., 1973; Graves, 1973
; Inoue et al., 1993
; Knowles & McCauley, 1997
; Seechurn et al., 1990
; Zhang et al., 1993
). The antigenic and molecular relationships between these two viruses suggest that CVB5 crossed the species barrier from humans to pigs at some time between 1945 and 1965, when it was first identified as a porcine pathogen, and has since continued to adapt to the new host (Zhang et al., 1999
).
The progenitor virus CVB5 uses the coxsackieadenovirus receptor (CAR) as primary receptor and decay-accelerating factor (DAF; CD55) as co-receptor (Bergelson et al., 1997; Martino et al., 2000
; Shafren et al., 1995
; Spiller et al., 2000
). Several other human enteroviruses, including CVB serotypes 1 and 3 and serotypes of echovirus, enterovirus 70 and coxsackievirus A21, have also been reported to bind human DAF (Bergelson et al., 1994
; Karnauchow et al., 1996
; Shafren et al., 1997
; Ward et al., 1994
). Although these viruses belong to the same family, different viruses bind to different sites on DAF, suggesting independent evolution of DAF binding (Bergelson et al., 1994
, 1995
; Clarkson et al., 1995
; Karnauchow et al., 1998
; Shafren et al., 1995
, 1997
). It was therefore of interest to investigate whether SVDV had retained receptor-binding properties after crossing to pigs.
Sequences for many different SVDV strains isolated over the last 3540 years are now available and sequence comparisons allow a unique insight into the selective pressures required for maintaining consensus sequences in the capsid of this virus. SVDV has been subdivided into four groups, AD, based on sequence and antigenic-epitope divergence from CVB5 (Brocchi et al., 1997). Receptor usage by SVDV is poorly defined. A single report found that mAbs against human DAF or human CAR each decreased the capacity of SVDV strain UK 27/72 to infect human HeLa cells (Martino et al., 2000
). UK 27/72 is a member of group B and is representative of SVDV isolates from early outbreaks of disease that are highly similar to CVB5, whilst isolates obtained in the early 1990s belong to groups C and D and have far less similarity to CVB5. No data currently exist regarding receptor usage of the isolates from more recent outbreaks.
Here, we compare the ability of progenitor CVB5 and SVDV isolates from early and recent outbreaks to utilize DAF and CAR for binding and infection of cells. The ability of specific antibodies raised against recombinant DAF and CAR, as well as pre-incubation of the virus with these recombinant proteins, to block infection of permissive cells was used to assess receptor usage. The role of human DAF in human and porcine virus infection was also assessed through radiolabelled virus binding and enhancement of infection following expression of human DAF on pig cells. The inability of these viruses to bind to pig DAF was also confirmed.
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METHODS |
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The SVDV isolates used in this work were: UK 27/72 (UK'72; GenBank accession no. X54521) as reference isolate, provided by the Institute of Animal Health (IAH, Pirbright, UK); IT/1/'66 (It'66; GenBank accession no. Y14464), from the first SVDV outbreak in 1966; isolates R1072 (Borrego et al., 2002) and R1120 (GenBank accession no. Y14474), from two independent outbreaks in the early 1990s, provided by E. Brocchi, IZSLE, Brescia, Italy; and the SPA/1/'93 isolate (SPA'93; GenBank accession no. AY157625), from the most recent Spanish SVDV outbreak (Espuña et al., 1993
; Jiménez-Clavero et al., 1998
). The CVB5 reference isolate (Faulkner strain; GenBank accession no. AF114383) was provided by A. Tenorio, Enterovirus Laboratory, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain. Low passage number is crucial to maintaining the integrity of virus receptor usage from the initial isolate and was kept to a minimum: R1120 and R1072 at three to four passages on IB-RS-2 cells, SPA'93 and UK'72 at seven to eight passages on IB-RS-2 cells, It'66 at eight to ten passages on IB-RS-2 cells and CVB5 (not including passage history at ATCC) at four to five passages on BGM cells.
Binding assays.
Viruses were labelled metabolically with 0·42 mCi (15·5 MBq) [35S]Cys/Met (Trans-Label; ICN) as described previously (Spiller et al., 2000). Following removal of cell debris by centrifugation (3000 g), labelled viruses in the supernatant were separated from unincorporated radiolabel by centrifugation (125 000 g) through a 30 % sucrose cushion in PBS (pH 7·4). Pelleted viruses were resuspended in serum-free medium and particulate material was removed by centrifugation at 16 000 g for 10 min at 4 °C and stored frozen at 70 °C in aliquots until used. A 50 µl aliquot of each virus preparation (108 TCID50 ml1), containing the following amounts of incorporated 35S label, was used for each sample: SVDV It'66 (80 349 c.p.m.), SVDV UK'72 (52 726 c.p.m.), SVDV R1072 (47 790 c.p.m.), SVDV R1120 (59 829 c.p.m.), SVDV SPA'93 (41 747 c.p.m.) and CVB5 (45 494 c.p.m.). For each sample (repeated in triplicate), 107 EDTA-disaggregated cells were pelleted and resuspended in 50 µl serum-free medium containing radiolabelled virus and incubated on ice for 2 h. Unbound virus was removed by three 0·25 ml washes (1000 g, 5 min) in ice-cold, serum-free cell medium and bound virus was quantified by scintillation counting. Statistical analysis was performed by using one-way ANOVA followed by a Tukey test post hoc (GraphPad Software).
Virus-infection assays.
Duplicate 10x dilutions of virus stocks were incubated with confluent monolayers of HeLa, IB-RS-2 or transfected cell lines grown in 96-well plates, in serum-free DMEM supplemented with antibiotics, for 4872 h in a CO2 incubator at 37 °C until development of cytopathic effect (CPE) was observed. Virus titre was defined as the reciprocal of the highest virus dilution able to produce detectable CPE in this assay.
Antibody-blocking assays.
IB-RS-2 or HeLa cells were seeded into 96-well plates as for the virus-infection assays described above. For antibody blocking, 30 min prior to addition of a 10-fold dilution series of each virus, cells were incubated with 50 µg ml1 of rabbit polyclonal anti-human DAF, anti-pig DAF or anti-CAR IgG (which cross-reacts with both species; Spiller et al., 2002). Rabbit IgG was dialysed into PBS and filter-sterilized prior to addition to cells. Virus infections with stocks of known virus titrations performed in the presence of anti-DAF or anti-CAR were compared with those performed in the absence of antibody.
Blocking experiments with soluble receptors.
IB-RS-2 cells were seeded into 96-well plates and allowed to grow to 90 % confluence. Prior to addition to cells, a 10-fold dilution series was made from each virus stock in serum-free medium and an equal volume of serum-free medium or serum-free medium containing 5 µM sterile, recombinant human (h) DAF-Fc or hCAR-Fc fusion proteins was added and incubated on ice for 30 min. Details of the construction, characterization and purification of soluble recombinant DAF or CAR fused with the Fc region of human IgG are provided elsewhere (Yanagawa et al., 2003, 2004
). Infections were observed for CPE daily, but were fixed with 1 % formaldehyde and stained with 0·1 % crystal violet in PBS to observe residual cells at 72 h post-infection (p.i.).
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RESULTS |
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DISCUSSION |
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Given that pigs are the natural host for SVDV, we then expanded our investigations of SVDV infection to the permissive pig-cell line IB-RS-2. The ability of CAR to mediate infection of all SVDV isolates was confirmed through complete inhibition of viral infection by pre-incubation of pig cells with antiserum recognizing pig CAR, or pre-incubation of SVDV isolates with recombinant human CAR (Table 1 and 2). We found the SVDV isolate UK'72 to be the least sensitive to inhibition by recombinant human CAR, whereas our isolate from a much more recent outbreak, SPA'93, was the most sensitive (Table 2
). Even though IB-RS-2 cells expressed pig DAF (Fig. 1
), pre-incubation with rabbit polyclonal anti-pig DAF did not inhibit infection (Table 1
). Furthermore, no binding of any radiolabelled SVDV isolate to CHO cells expressing pig DAF (expressed 135-fold times greater than levels on IB-RS-2 cells) was observed above binding to CHO control cells. However, it is unlikely that human DAF-utilizing viruses would evolve to use the pig DAF homologue, due to large structural differences between these proteins. Pig DAF is comprised of only three complement-control (CCP) domains, whereas human, rat and mouse DAF contain four CCP domains (Pérez de la Lastra et al., 2000
). Sequence comparison amongst these proteins indicates that it is the homologue of the fourth DAF CCP domain that is missing and, as a result, pig DAF regulates human complement activation poorly and does not regulate pig complement at all (Pérez de la Lastra et al., 2000
).
We still wished to assess whether the more recent SVDV isolates retained the ability to bind and utilize human DAF as a receptor. Whilst we found that pre-incubation of recombinant human DAF with CVB5 resulted in a reduced infection of pig cells, recombinant human DAF was unable to inhibit infection of any of the SVDV isolates. Although this inhibition cannot be due to competition between soluble and surface receptors, it is probably caused by steric hindrance, given the close proximity between DAF- and CAR-binding sites on the capsid, similar to our previous report showing that hDAF-Fc inhibited CVB3 infection of mice (Yanagawa et al., 2003). Transfection of the pig cells with human DAF was found to increase the titre of early SVDV isolates, but no significant effect was noted for infection of these cells by the isolates from more recent outbreaks (Table 3
). Similar results were observed for the binding of radiolabelled CVB5 and SVDV isolates to IB-RS-2 cells expressing human DAF relative to binding to control cells: a big increase in binding for CVB5, a small increase in binding for SVDV isolates from early outbreaks and no increase in binding for SVDV isolates from more recent outbreaks. We conclude, therefore, that as SVDV has evolved in its new host, the capsid structures responsible for human DAF binding have been lost, due to the absence of this co-receptor or any close pig homologues.
Many recent studies have focused on identifying these capsid structures and it is possible that comparison of the capsid sequences between CVB5, SVDV and these other viruses may help to identify essential and non-essential residues in these areas (Fig. 2). These comparisons are greatly enhanced by the recent X-ray crystallography solution of the UK'72 isolate by Fry et al. (2003)
and of the SPA'93 isolate by Verdaguer et al. (2003)
. Whilst the structures of SVDV complexed with CAR or DAF have not been solved directly, conclusions can be drawn by comparing the cryo-electron microscopy analysis of CVB3CAR and echovirus 7DAF complexes reported by He et al. (2001
, 2002)
. The capsid sequences surrounding the larger parts of these footprints have been aligned for CVB3, echovirus 7, CVB5, early SVDV isolates and recent SVDV isolates in Fig. 2
, which shows significant similarity between all of the viruses. Several changes to capsid sequence are shown within and surrounding these footprints by comparing CVB5 with SVDV and comparing SVDV isolates from early and more recent outbreaks; however, a more detailed comparison of sequences for the entire capsid-coding region, without emphasis on the receptor-binding sites, can be found in the paper by Verdaguer et al. (2003)
. The CVB3 capsid sequences identified as binding to CAR were found primarily in the VP1 capsid protein, with some contribution of the hypervariable puff region in VP2. The echovirus 7 sequences identified as binding to DAF have also been mapped to the VP2 puff region (region A), although two other, smaller, DAF-binding regions (B and C) have been found in VP3, which include the hypervariable VP3 knob region. Although the initial sequence comparison found that recent SVDV isolates have more DAF-binding contact residues conserved with CVB5 than the early SVDV isolates, there are many changes in the intervening capsid sequence that could result in the loss of DAF binding. Alignment of the echovirus 7 DAF-binding sequence IKV in the VP3 knob region was not exact; however, a GKV sequence is located at almost the same position in early SVDV isolates and this sequence is altered in recent SVDV isolates to GKE, which could also be responsible for the loss of DAF binding. Four sequence alterations that were conserved between all SVDV isolates were not found in CVB5: T2151I, S2161T and I2177V in the VP2 puff region, and Q1091Y in the CAR-binding region of VP1. There were also four sequence alterations that were found in all SVDV isolates other than It'66: Q2143P, N2148K and Q2163E in the VP2 puff and N1210S in the VP1 CAR-binding region. Four capsid alterations found only in SVDV isolates from recent outbreaks included N2153S and V2154T in the VP2 puff, V3062E in the VP3 knob and Q/R1258E in the VP1 CAR-binding region, which may be responsible for the loss of DAF binding. A unique change found only in SPA'93, A2165D, was also found in the VP2 puff region, and it is tempting to speculate that this may be responsible for the high sensitivity of this isolate to inhibition by recombinant CAR. However, it is important to note that cryo-electron microscopy analysis has never been used to solve DAFCVB complex structures and it is possible that the DAF-binding region of CVB capsids is distinct from that identified for echovirus 7. Whilst the cryo-electron microscopy analysis of echovirus 7 found that DAF bound along the twofold axis of symmetry, Stuart et al. (2002)
reported that echovirus 11 bound to the platform surrounding the fivefold axis of symmetry on the capsid. This latter study relied on modelling capsid mutations in echovirus 11 strains that had lost DAF binding; they also identified a contribution of the VP2 puff region, which has been included in Fig. 2
. However, for the most part, the DAF-binding regions of both echovirus 7 and 11 represent the areas of greatest sequence divergence when compared with SVDV and CVB, reinforcing the hypothesis that DAF probably binds to a different area of the CVB and early SVDV isolate capsids compared with echoviruses. Whether the loss of DAF binding or possible increase in CAR binding is due to any one, or a combination of, the above identified capsid sequence alterations will require extensive studies utilizing site-directed mutagenesis of infectious cDNA constructs.
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ACKNOWLEDGEMENTS |
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Received 6 October 2004;
accepted 3 February 2005.
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