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Journal of Virology, October 1999, p. 8496-8502, Vol. 73, No. 10
Department of Veterinary
Pathology1 and Department of Veterinary
Clinical Sciences and Animal Husbandry,2
University of Liverpool, Veterinary Teaching Hospital, Leahurst, Neston
CH64 7TE, United Kingdom
Received 24 May 1999/Accepted 12 July 1999
In order to map linear B-cell (LBC) epitopes in the major capsid
protein of feline calicivirus (FCV), an expression library containing
random, short (100- to 200-bp) fragments of the FCV F9 capsid gene was
constructed. Analysis of this library showed it to be representative of
the region of the capsid gene that encodes the mature capsid protein.
The library was screened by using polyclonal antisera from a cat that
had been challenged experimentally with F9 to identify immunoreactive
clones containing LBC epitopes. Twenty-six clones that reacted
positively to feline antisera in immunoblots were identified.
FCV-derived sequence from these clones mapped to a region of the capsid
that spanned 126 amino acids and included variable regions C and E. An
overlapping set of biotinylated peptides corresponding to this region
was used to further map LBC epitopes by using F9 antisera. Four
principal regions of reactivity were identified. Two fell within the
hypervariable region at the 5' end of region E (amino acids [aa] 445 to 451 [antigenic site {ags} 2] and aa 451 to 457 [ags 3]).
However, the other two were in conserved regions (aa 415 to 421 [ags
1; region D] and aa 475 to 479 [ags 4; central region E]). The
reactivity of the peptide set with antisera from 11 other cats infected
with a range of FCV isolates was also determined. Ten of 11 antisera reacted to conserved ags 4, suggesting that this region may be useful
for future recombinant vaccine design.
Feline calicivirus (FCV) is an
important acute, oral and respiratory pathogen of domestic cats
(13) and belongs to the family Caliciviridae
(6). It contains a single-stranded, positive-sense RNA
genome of approximately 7.7 kb that contains three open reading frames
(ORFs) (Fig. 1A) (2, 16, 30, 32, 35,
50, 53). ORF1 is located at the 5' end of the genome and codes
for the nonstructural proteins. ORF3 encodes a putative minor
structural protein. ORF2 encodes the major capsid protein and is
divided into six regions designated A to F (Fig. 1B). Region A is
cleaved to release the mature capsid protein (3). Regions B,
D, and F are relatively conserved between FCV isolates, whereas regions C and E are variable between isolates (31, 46-48). Region E
has been further divided into 5' and 3' hypervariable regions (HVRs) separated by a conserved central domain (Fig. 1B) (48).
Whereas isolates of FCV can often be distinguished from each other both antigenically (8, 24, 28, 40) and by sequence analysis (14, 16), FCVs are currently considered to belong to a
single serotype (40) and a single genotype (14,
16).
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
The Capsid Gene of Feline Calicivirus Contains
Linear B-Cell Epitopes in both Variable and Conserved Regions
ABSTRACT
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
View larger version (32K):
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FIG. 1.
Summary of FCV structure. (A) The FCV genome contains
three ORFs. (B) ORF2 encodes the major capsid protein, which is divided
into conserved regions (regions B, D, and F) and variable regions
(regions C and E, including 5' HVR and 3' HVR). (C) Antigenic regions
previously identified: i, aa 408 to 517 (18);
ii, aa 422 to 458 (29); iii, aa 441 to
455 (55); iv, aa 493 to 494 (55);
v, aa 420 to 529 (33). (D) Map of region ags 1, 2, 3, and 4 identified in this study is shown. All amino acids are
numbered in accordance with the published FCV F9 capsid sequence
(3).
Following recovery from clinical disease, cats may develop a persistent, inapparent infection with FCV, and such carriers represent a reservoir of infection for susceptible animals (41, 58). Vaccination against FCV is available (13). The vaccines used are based on whole virus, often an isolate called F9. Although generally effective at preventing clinical disease, cats may develop subclinical and persistent infections under the protection of vaccine-induced immunity (10, 12). Despite vaccination, the prevalence of FCV within the cat population has remained high (5, 19), at levels similar to those reported prior to its introduction (59). Vaccine failures have also been reported. In the majority of cases, these are associated with field virus (8, 42), and it is likely that due to the antigenic variability of FCVs, current vaccines will not induce protection against all field isolates (14, 27). However, vaccine-derived virus has also been implicated as the cause of disease in some vaccine failures (8, 42).
Following infection with FCV, serum virus-neutralizing (VN) antibodies develop by approximately 7 days postinfection (21, 25). The levels of such VN antibodies correlate well with protection against homologous challenge (39). There is also production of immunoglobulin G (IgG)- and IgA-associated mucosal immunity (25) and serum immunofluorescence, complement fixation, complement fixation inhibition, and agar gel precipitation antibodies (15, 34, 57). Although major histocompatibility complex-restricted cytotoxic activity of peripheral blood T lymphocytes has been demonstrated in vaccinated cats, the significance of cytotoxic T lymphocytes to FCV protection is not known (51).
Attempts to characterize the antigenically important regions of FCV are summarized in Fig. 1C. A recombinant peptide corresponding to amino acids (aa) 408 to 517 of FCV F9 induced the formation of neutralizing polyclonal antisera in rabbits, and cats vaccinated with F9 produced a polyclonal antisera that reacted to this peptide (18). The peptide corresponding to aa 422 to 458 contained the epitopes for two neutralizing mouse monoclonal antibodies (N-MAbs) (29). Amino acids 441 to 445 and 493 to 494 are critical to the formation of four linear and three conformational epitopes, respectively (55) (see also Fig. 2). Finally, aa 420 to 529, when transferred from one FCV isolate to another, conveyed neutralization characteristics of the donor virus to the recipient (33). No neutralizing epitopes have currently been mapped in conserved regions of the FCV genome, although one N-MAb to a conformational FCV capsid epitope neutralized all FCV isolates tested (52).
Despite these previous studies, no methodical search has been carried out for linear B-cell (LBC) epitopes recognized by feline antibodies in the FCV capsid. The aim of this study was to construct an expression library containing random, short fragments of an FCV capsid gene, suitable for screening with antisera from an experimentally infected cat. The isolate chosen to make the library (F9) is frequently incorporated in FCV vaccines. By using this library, 26 immunoreactive clones were mapped within the capsid. Subsequent fine mapping of LBC epitopes within this region was performed by using an overlapping set of 9-mer peptides.
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MATERIALS AND METHODS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Construction of plasmid p1BSF9. The construction of pF9VAC, which contains the mature capsid protein coding region of FCV F9 (nucleotides 5686 to 7329 [2]), has been described elsewhere (17) and was kindly provided by M. Glenn. To facilitate subsequent manipulation, the F9 capsid gene was excised from pF9VAC and cloned into pBluescript (Stratagene). One clone, designated p1BSF9, was sequenced to confirm that it contained regions B to F of the F9 capsid (nucleotides 5686 to 7329 of the published F9 sequence [2]).
Construction of lambda expression library 
F9CAP.p1.
Random overlapping fragments of p1BSF9 of 100 to 200 bp were generated
by bovine pancreatic DNase I (Boehringer Mannheim) digestion using
standard protocols (45). The correct degree of digestion was
achieved by serial 10-fold dilutions of DNase I, and all reactions were
performed for 1 min at 14°C. Products of the correct size range were
gel purified, blunt ended with the Klenow fragment of DNA polymerase I
(Stratagene), and ligated to EcoRI linkers
(5'-CCGGAATTCCGG-3'; Stratagene) by using manufacturers' and standard protocols (45). The products of ligation were
digested with EcoRI. Fragments of the appropriate size were
gel purified, ligated into the lambda gt11 expression system, and
packaged in accordance with the manufacturer's instructions (lambda
gt11 system and Packagene; Promega).
F9CAP. Initial screening of
F9CAP was performed by blue-white plaque selection. The library was
amplified and aliquoted to produce working stocks designated F9CAP.p1, which were used in all subsequent experiments.
Characterization of insert size within F9CAP.
The size of
individual inserts within nine randomly picked plaques from F9CAP
was determined by PCR amplification across the gt11 cloning site
using lambda gt11 forward and reverse primers (Promega) in accordance
with the manufacturer's instructions.
Characterization of insert origin within F9CAP.p1.
In
situ hybridization using three probes corresponding to the 5', middle,
and 3' regions of the mature FCV capsid coding region was used to
confirm that F9CAP.p1 contained DNA of FCV origin and that the
library was representative of the entire coding region.
-32P]dCTP (Prime-it RmT random
primer labelling kit; Stratagene), and purified with Sephadex G50
columns (ProbeQuant G-50 microcolumns; Pharmacia Biotech) in accordance
with the manufacturers' instructions.
Target DNA was prepared by plating F9CAP.p1 in order to produce
approximately 300 discrete lambda plaques on a standard 90-mm-diameter petri dish. Plaque blots were prepared in triplicate with a Hybond-N nylon membrane (Amersham). Blots were processed and hybridized essentially in accordance with the manufacturer's instructions, using
stringent wash conditions.
Antisera. Antisera from specific-pathogen-free cats were prepared as described previously (9), using FCV isolates F9 (1), LS015 and LS027 (25), and Bu13 (7), three commercial vaccine viruses based on F9 (VacA, VacB, and VacC), and a previously undescribed American field isolate (FCVus1).
Immunological screening of F9CAP.p1.
Hybond-N nylon
membrane filters (Amersham) were soaked in 10 mM
isopropyl-
-D-thiogalactopyranoside (IPTG; Sigma) and air dried. All wash stages were performed at room temperature with gentle
agitation unless otherwise stated.
F9CAP.p1 were plated in 90-mm-diameter
petri dishes by using standard protocols (Promega). As negative
controls, antiserum against F9 was also used to screen recombinant
lambda gt11 containing a non-FCV-derived insert as supplied by the
manufacturer (Promega). Plates were incubated at 42°C for
approximately 4 h until plaques were just visible, overlaid with
the IPTG-impregnated membranes, and incubated overnight at 4°C. The
membranes were removed from the plates and washed twice in
phosphate-buffered saline (PBS; 154 mM NaCl, 3 mM KCl, 9 mM
Na2HPO4, 1.65 mM
KH2PO4) for 5 min. Membranes were blocked for
1 h in 1% (wt/vol) bovine serum albumin (BSA; Sigma) in PBS and
washed twice in 0.05% Tween 20 (Sigma) in PBS (PBS-T).
Anti-F9 serum from cat 1 was diluted 1:200 in 0.5% (wt/vol) BSA in
PBS. Membranes were incubated with 10 ml of this primary antiserum for
1 h at 37°C. After the membranes were washed three times in
PBS-T for 5 min per wash, they were incubated in 10 ml of mouse
monoclonal anti-cat IgG biotin conjugate (Sigma) diluted 1:6,000 in
PBS-T containing 0.5% BSA (wt/vol). The membranes were washed three
times in PBS-T for 5 min per wash and incubated for 10 min in
extravidin peroxidase (Sigma) diluted 1:2,000 in 0.5% (wt/vol) BSA in
PBS-T. The membranes were washed for 5 min, twice in PBS-T and twice in
PBS, and incubated at room temperature with gentle agitation with fresh
3,3'-diaminobenzadine tetrahydrochloride (DAB; Sigma) prepared in
accordance with the manufacturer's instructions until plaques became
visible. The reaction was terminated by washing the membranes twice in
tap water and air dried. Positive plaques were stored in 1 ml of phage
buffer and subjected to two further rounds of immunological screening
prior to sequencing.
Sequencing of DNA from positive plaques. For sequencing, DNA from reactive plaques was amplified by PCR. Briefly, 10 µl of phage buffer containing each stored plaque was added directly to a 90-µl PCR mix containing 5 U of Taq DNA polymerase and 1× PCR buffer (Advanced Biotechnologies), 100 µM each deoxynucleoside triphosphate, and 200 nM each of primer gt1 (5'-CGGTTTCCATATGGGGATTGGTGGCG-3') and gt2 (5'-CGCGAAATACGGGCAGACATGGCCTGC-3') (Kings College, London, United Kingdom). Thermal cycling conditions consisted of 95°C for 2 min, followed by 40 cycles of 95°C (1 min), 50°C (1 min), and 72°C (3 min). A final extension was performed at 72°C for 5 min. A negative control of water was processed simultaneously. Amplicons were purified (Wizard PCR prep DNA purification system; Promega) and sequenced (Prism big dye terminator cycle sequencing ready reaction kit; Perkin-Elmer ABI 377) in accordance with the manufacturers' instructions.
Peptide mapping.
To span the 126-aa region identified
by screening F9CAP.p1, overlapping 9-mer peptides
corresponding to this region were synthesized, each peptide offset from
the next one by 2 aa. Peptides were made simultaneously on derivatized
polyethylene pins (Chiron Technologies, Clayton, Australia), cleaved,
and supplied as lyophilized powders (peptides 3 to 62). Peptides were
N-terminally biotinylated and included a 4-aa spacer arm (SGSG).
Negative controls consisted of two similar peptides not based on FCV
sequence (peptides 1 and 2), and they were supplied by the
manufacturer. Two FCV-based peptides that map outside the 126-aa region
were also synthesized (RHFDFNQET and QSKIVVFQD; peptides 63 and 64, respectively). The peptide set was screened by using a solid-phase
immunoassay in accordance with the manufacturer's protocol. Briefly, a
1:200 dilution of antisera from FCV-challenged cats was used to detect LBC epitopes. Bound feline IgG was detected by using a 1:1,000 dilution
of peroxidase-labelled goat anti-feline IgG (Kirkegaard & Perry
Laboratories, Inc.) and ABTS
[2,2'-azinobis(3-ethylbenzthiazolinesulfonic acid)] (Sigma). Optical
density (OD) values (405 nm) for each peptide were determined in
duplicate, averaged, and corrected by subtracting the average of the
two negative control peptides.
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RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Predicted amino acid sequence of the mature capsid coding region
derived from p1BSF9 is shown in Fig. 2.
Results of PCR analysis of nine randomly picked plaques from
F9CAP.p1 are shown in Fig. 3. Amplicon sizes range from
approximately 180 to 310 bp, which is equivalent to lambda gt11 insert
sizes of 90 to 220 bp (PCR amplification across the cloning site of
gt11 adds approximately 90 nucleotides of vector-derived DNA to the
insert). Probes representing the 5', middle, and 3' regions of the F9
capsid gene each reacted positively with approximately 10% of plaques
in F9CAP.p1 (data not shown). This is in broad agreement with the
proportion of p1BSF9 that was FCV derived and suggested that
F9CAP.p1 was equally representative of the mature capsid-coding
region.
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and 
indicate sites of mutations that disrupt LBC and
conformational B-cell epitopes, respectively, in FCV F4
(Immunoscreening of F9CAP.p1 with anti-F9 serum from cat 1 identified
approximately 10 immunoreactive plaques per 2,000 PFU. The antiserum
did not react positively to any plaques in the lambda negative control.
Sequence was obtained from 26 of the immunoreactive plaques after two
further rounds of plaque purification. Reactive clones covered 126 aa
between aa 382 and aa 507 and included sequence from the 3' end of
region B to the 5' end of the 3' HVR of region E (Fig.
2). Several clones did not overlap each
other, suggesting that there were at least two regions containing LBC
epitopes. No immunoreactive clones were identified outside this region, despite several attempts using duplicate blots and attempting to select
immunoreactive clones that did not hybridize in a Southern blot to a
probe containing sequence from the 5' HVR (data not presented).
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X174
molecular weight markers (Boehringer Mannheim) (with values in base
pairs indicated on the right). Amplicon sizes range from approximately
180 to 310 bp.
Results of screening the overlapping peptide set using antisera from
cats infected with different FCV isolates are shown in Fig.
4 and 5.
Anti-F9 serum from cat 1 demonstrated four putative LBC
epitope-containing antigenic sites (ags) identified as GIPDGWP (aa 415 to 421; peptides 19 and 20, ags 1), ITTATGY (aa 445 to 451; peptides 34 and 35, ags 2), YDTADII (aa 451 to 457; peptides 37 and 38, ags 3), and
AWGDK (aa 475 to 479; peptides 48 to 50, ags 4), with the strongest
reactivity against region ags 3. ags 1 was located in conserved region
D, ags 2 and ags 3 were located in the 5' HVR of region E, and ags 4 was located in the conserved central part of region E (Fig. 1D and 2).
All immunoreactive clones identified in F9CAP.p1 encoded at least
one of the ags, either ags 1, ags 2, ags 3, or ags 4 (data not
presented).
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When anti-F9 serum from cat 2 was used, the strongest reactivity was to both variable region ags 2 and ags 3. However, ags 2 was 2 aa further towards the NH2 terminus of the protein than that with the antiserum from cat 1 (NDITTAT; aa 443 to 449). There was also some activity against region ags 1, but surprisingly, there was no detectable reactivity in region ags 4.
Antisera raised to vaccine viruses (VacA, VacB, and VacC) reacted most with variable ags 3 and conserved ags 4. Reactivity to other regions was less apparent, only exceeding 25% of maximum for VacA antiserum in ags 2.
Antisera from the remaining cats infected with the field isolates reacted consistently with peptides corresponding to conserved ags 4. For antiserum against LS015, F65, and LS027, reactivity was strongest to this region. However, for the remaining antisera (FCVus1 and Bu13), reactivity was as strong or stronger to region ags 3. Overall, reactivity to ags 1 and ags 2 was less apparent.
A low-level reactivity was frequently observed with peptides 3 and 4, but this was also noted with the negative control antisera.
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DISCUSSION |
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Discussion
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It is important to identify those regions of viruses that closely interact with host proteins since these regions determine the pathogenesis and clearance of the virus. For caliciviruses, such regions are largely unknown, although in FCV, current evidence suggests that the 5' HVR of region E contains the immunodominant regions of the capsid (18, 29, 49, 55, 56). However, these studies either have used nonfeline antibodies (29, 49, 55) or have not analyzed the whole of the FCV capsid coding region (18). In this study, we have carried out a systematic search of the mature FCV capsid for LBC epitopes with feline antisera. Using an expression library, we have shown that the only immunoreactive region of the capsid appears to be confined to 126 aa spanning variable regions C to E. Within these regions, we have demonstrated the presence of at least four putative LBC epitopes by using an overlapping peptide library. Two of these regions (ags 2 and ags 3) map close to each other in the 5' HVR. However, the other two (ags 1 and ags 4) map to region D and the middle of region E, respectively, in regions of the FCV capsid that are considered to be conserved on the basis of sequence analysis.
In a previous study using mouse N-MAb escape mutants (55), four neutralizing LBC epitopes clustered in the 5' HVR of FCV isolate F4 were identified (Fig. 2). Mutations that disrupt these epitopes map very close to the ags 2 and ags 3 identified in our study (55). This confirms the significance of the 5' HVR as a major antigenic determinant in FCV and as an important target for neutralizing antibodies. Indeed, we have previously shown that a mutation at aa 449 of the capsid allows F9 to escape neutralization by the N-MAb IG9 (4) (data not shown).
In addition to these variable epitopes, our study also identified epitopes in conserved regions on either side the 5' HVR which have not been reported in previous epitope mapping studies. Such conserved B-cell epitopes may ultimately prove to be good candidates for future vaccine design, although the neutralizing ability of antibodies targeting such regions has not yet been determined. Neutralizing immune responses targeting conserved epitopes should in theory be broadly cross-reactive and reduce the risk of viral escape mutant formation. This may be particularly true for the ags 4 identified in this study, since all the cats that were infected with non-F9 FCV isolates also produced antibodies that reacted with this conserved region.
Although we have identified conserved antigenic sites in this study, one of the two cats infected with F9 failed to produce a detectable antibody response to ags 4, and the majority of cats failed to produce detectable antibodies to conserved ags 1. This may suggest that in the isolates used to produce antisera in this study, there may have been minor sequence variability in these otherwise conserved regions, sufficient to disrupt the epitopes. Unfortunately, capsid sequence is not available for all the isolates used in this study. However, in comparisons of the 22 full capsid sequences available to the authors (16), whereas ags 4 was completely conserved, ags 1 contained three sequences. Nineteen isolates had the sequence GIPDGWPA as in F9, one contained a single substitution (GIQDGWP; FCV 2280), and one contained an insertion and a substitution (GIPDQVWP; FCV LLK). This suggests that ags 1 may tolerate more variability than ags 4 and explain why, in this study, ags 1 reactivity was less than that of ags 4.
Alternatively, there may be variability in the response of individual cats to conserved epitopes. This may simply reflect antibody titer differences between antisera used in this study such that if a higher concentration of antiserum had been used, antibodies to these conserved epitopes may have ultimately been detected. It is also possible that variability in antigen presentation associated with differences in histocompatability type may have meant that some cats responded less efficiently to these epitopes.
The failure of studies based on mouse MAbs to identify conserved epitopes may reflect the unnatural host in which MAbs against FCV were made. It is likely that as the natural host, cats infected with FCV (as in this study) are exposed to a greater repertoire of antigens than the mice used to produce MAbs and in which FCV is considered not to replicate. It is also likely that the populations of mice used to produce MAbs represent inbred populations, and, as such, the repertoire of antigens they respond to is likely to be more restricted. Since the earlier MAb studies selected N-MAbs (54); it is also possible that the conserved epitopes we have identified induce nonneutralizing antibodies. It will therefore be important to further characterize the antibodies that react with these conserved regions, as to their ability to neutralize FCV. The presence of shared and therefore possibly conserved epitopes targeted by neutralizing antibodies is suggested by the observed cross-reactivity between FCV antisera and heterologous FCV isolates in vitro which has led to the single serotype definition of FCVs (40). There has also been an N-MAb described which consistently neutralizes different FCV isolates in vitro and that reacts with a conformational epitope in the capsid (1D7) (52). However, attempts to map the 1D7 epitope have been unsuccessful, since it has not been possible to manufacture an escape mutant to this antibody, suggesting that this epitope forms part of an essential region of the FCV capsid (52).
In this study, no epitopes were mapped to other variable regions of the capsid, namely, region C and the 3' HVR. This suggests that if the observed variability in these regions is immunologically significant, then these regions may contain either conformational B-cell epitopes or T-cell epitopes. Indeed, the 3' HVR has been implicated in the formation of conformational epitopes using N-MAb escape mutants (55). Fine structural studies of the calicivirus capsid that would identify secondary conformational interactions between regions of primary capsid protein sequence have not yet been performed.
The colocalization of neutralizing B-cell epitopes with HVRs in viral capsid and envelope proteins is well recognized and is believed to reflect the presence of these domains on the surface of viral proteins. In the envelope gene (env) of human immunodeficiency virus type I, the third variable region contains B- and T-cell epitopes (20, 36, 44). In feline immunodeficiency virus, B-cell epitopes have also been mapped to HVRs in env (37). Equivalent regions have also been identified in HVRs of hepatitis C virus envelope glycoprotein (23, 60).
The inherent variability of such surface-expressed immunodominant regions has important implications to FCV antigenicity and neutralization by the host. First, it may explain the wide spectrum of related but slightly different antigenic profiles seen between FCV isolates (8, 9, 22, 24, 40). Second, FCV isolates have been shown to evolve both antigenically and by sequence analysis of the capsid HVRs in carrier cats, suggesting a possible role for virus evolution in the mechanism of FCV persistence (26, 43). Finally, since most FCV vaccines have traditionally been based upon a single isolate, often F9, it is probable that vaccine-induced antibodies that target variable regions will not protect equally against all FCV isolates (8, 9, 38). Indeed, FCV-related disease in vaccinated cats, although uncommon, is well described (8, 9, 38). Conventional polyvalent and subunit recombinant vaccines have been developed in an attempt to improve the cross-reactivity of vaccine-induced immunity (9, 11). In the latter case, a recombinant polypeptide containing regions C to E from five different FCV isolates was able to induce neutralizing antibodies and some protection from FCV challenge but did not alter the duration of FCV shedding compared to that in controls (11).
In conclusion, we have confirmed that the 5' HVR of capsid region E is an important immunodominant region of FCV, containing linear B-cell epitopes. However, we have for the first time mapped epitopes to more conserved regions of the FCV capsid. Although the importance of these epitopes to virus neutralization needs to be determined, such epitopes may account for the observed cross-reactivity between FCV strains and may lead to the development of improved recombinant vaccines capable of providing protection against all field isolates.
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ACKNOWLEDGMENTS |
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The authors are especially grateful to Ruth Ryvar for technical assistance.
We thank Margaret Hughes at the Liverpool School of Tropical Medicine for all sequencing and Barry Hodson from Chiron Technologies for assistance with peptide set design and handling. We are also very grateful to Satya Malik for help with the enzyme-linked immunosorbent assays.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Veterinary Pathology, University of Liverpool, Veterinary Teaching Hospital, Leahurst, Neston CH64 7TE, United Kingdom. Phone: 44 151 794 6017 or 6012. Fax: 44 151 794 6005. E-mail: alanrad{at}liv.ac.uk.
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REFERENCES |
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Abstract
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Materials and Methods
Results
Discussion
References
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Journal of Virology, October 1999, p. 8496-8502, Vol. 73, No. 10
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