Identification of Another B-Cell Epitope in the Type-Specific Region of Equine Herpesvirus 4 Glycoprotein G

Recently, a novel 12-mer B-cell epitope, MKNNPIYSEGSL, in thetype-specific region of equine herpesvirus 1 (EHV-1) glycoproteinG (gG) was identified and used as an antigen for enzyme-linkedimmunosorbent assay (Maeda et al., J. Clin. Microbiol. 42:1095-1098,2004). Although our prototype strain, TH20p, possesses two repeatsequences containing the B-cell epitope, the EHV-4 NS80567 strainhas two repeat sequences that are not identical. One repeatsequence stretch contained the B-cell epitope, while the othercontained the 11-mer, MKNNPVYSESL (underlining indicates a differentamino acid). In this study, heterogeneity of the type-specificregion was compared among Japanese EHV-4 isolates. The 11-merpeptide, MKNNPVYSESL, specifically reacted with sera from horsesnaturally infected with EHV-4 but not with sera from horsesexperimentally infected with EHV-4 TH20p. The 11-mer peptidemay be another B-cell epitope in the type-specific region.

INTRODUCTION

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Introduction

Equine herpesviruses 1 (EHV-1) and 4 (EHV-4) are causative agentsof equine rhinopneumonitis. Because both viruses are closelyrelated genetically and immunologically, distinguishing thetwo has been very difficult. Crabb et al. identified the type-specificregion at the C-terminal region of glycoprotein G (gG) and establishedan enzyme-linked immunosorbent assay (ELISA) using these regionsas antigens (1, 2). Our group also developed a type-specificELISA using Japanese isolates (8) and used the ELISA for epizootiologicaland immunological studies in Japan (5, 9).

Because the ELISA antigens were expressed in Escherichia colias fusion proteins, the purification was time-consuming. Therefore,the type-specific 12-mer B-cell epitope of EHV-4, MKNNPIYSEGSL,was identified and used as an ELISA antigen (4). Although theJapanese prototype strain, TH20p, has two repeat sequences containingthe 12-mer B-cell epitope in the type-specific region, NS80567has one stretch of repeat sequences containing the 12-mer B-cellepitope and another one containing similar, but not identical,amino acids, MKNNPVYSESL (underlining indicates a differentamino acid) (7).

In this study, the heterogeneity of the type-specific regionwas compared among Japanese EHV-4 field isolates. In addition,ELISA using the 11-mer peptide was also established and comparedwith that using our recently identified 12-mer B-cell epitope.

MATERIALS AND METHODS

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Materials and Methods

Viruses and cells. Twenty-one EHV-4 field isolates and two laboratory strains wereused. The H45 strain was isolated from an aborted fetus in Japanand has been used as the Japanese prototype strain of EHV-4(6). The TH20p strain was plaque purified from strain TH20,isolated from a colt suffering from respiratory disease (3).The other 21 strains were isolated between 1981 and 1991 andwere propagated in fetal horse kidney (FHK) cells. Field isolateswere used in the third passage and were never plaque purified.The 19 strains were isolated from foals with respiratory disease;91c1 was from an aborted fetus, and 88c162 was from horses withoutany clinical signs. Two pairs of isolates, 88c160 and 88c162as well as 88c180 and 88C186, were simultaneously isolated fromdifferent horses from the same farm and are therefore consideredepidemiologically related.

DNA extraction. Twenty-one field isolates and the TH20 and H45 strains wereused for DNA analyses. Viruses were propagated at low multiplicitiesof infection in FHK cells. Infected cells were treated withl% sodium dodecyl sulfate and proteinase K (0.1 mg/ml) in asolution containing 0.1 M Tris-HCl, 0.1 M NaCl, 5 mM EDTA (pH9.0) at 37°C overnight. Following DNA extraction with phenoland chloroform-isoamylalcohol (24:1), ethanol-precipitated DNAwas dissolved in water.

Amplification of the type-specific regions. The type-specific regions were amplified from extracted DNAof field isolates by PCR using two primers, gG4P-F and gG4P-R(8). Amplified samples were separated by electrophoresis on1 to 2% agarose gels. Gels were stained with ethidium bromideand analyzed.

Nucleotide sequences. The amplified fragments were cloned into the pCRII vector (Invitrogen).The plasmids were analyzed by the ABI sequence analyzer. Nucleotidesequences were obtained for three independently cloned plasmids.

Synthetic peptides. Two peptides, G1 (MKNNPIYSEGSL) and G13 (MKNNPVYSE-SL), weresynthesized using the ACT 350 multiple peptide synthesizer (AdvancedChemTech) according to the manufacturer's protocol.

ELISA. Synthetic peptides were diluted to 10 µg/ml in phosphate-bufferedsaline (PBS), and 100 µl of the diluted peptides was addedto each well (Maxisorp; Nunc). As a control, PBS without peptidewas added to some wells. After incubation at 4°C overnight,the peptide-coated wells were washed three times with PBS andthen were incubated with 200 µl of 2% gelatin in PBS (enzymeimmunoassay grade; Bio-Rad) at room temperature for 1 h. Thewells were washed with PBS containing 0.05% Tween 20 (PBS-T),and 100 µl of sera diluted with PBS-T containing 1% gelatinwas added to the wells. After incubation at room temperaturefor 2 h, the wells were washed and incubated with 100 µlof diluted peroxidase-conjugated anti-horse immunoglobulin (EYLaboratories) at room temperature for 1 h. Following the washwith PBS-T and PBS, 0.05 mg of 2,2'-azino-di(3-ethlbenzothiazolinesulfonate)/ml was added to each well. After 30 min, the absorbancewas determined by using a spectrophotometer with a 405-nm filter.

Sera. Sera were collected at various times from six foals that wereintranasally inoculated with EHV-4 strain TH20p. From 1988 to1989, field sera were collected during the acute and convalescentphases from foals or yearlings with pyrexia due to EHV-4 infection.We also isolated EHV-4 from nasal swab samples from these horsesduring the acute phase.

RESULTS

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Results

Comparison of the type-specific region by PCR. The type-specific regions of 21 EHV-4 field isolates and twolaboratory strains, TH20p and H45, were amplified by PCR usingprimers gG4P-F and gG4P-R. As expected, approximately 300-bpfragments were amplified from 12 isolates and TH20p. However,approximately 370- and 450-bp fragments were detected from theother nine field isolates and H45, respectively (Fig. 1A).

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FIG. 1. PCR and amino acid sequences of the type-specific region of EHV-4 field isolates. (A) The type-specific regions were amplified from 23 EHV-4 isolates by PCR using primers gG4P-F and gG4P-R. (B) Amino acid sequences deduced from the nucleotide sequences of the type-specific regions of TH20p, 87c34, H45, and NS80567 (7) were compared. Bold letters and letters shown by underlines show repeat sequences containing G1 and G13, respectively.

Comparison of amino acid sequences in the type-specific region. Nucleotide sequences of three representative EHV-4 strains werecompared. The results showed that the size of the type-specificregions of TH20p, 87c34, and H45 were 295, 367, and 449 bp,respectively, and the number of repeat sequences was different(data not shown). Next, the amino acid sequences deduced fromnucleotide sequences were compared (Fig. 1B). Interestingly,although TH20p had two repeat sequences that were identical,87c34 and H45 had two kinds of repeat sequences. One containedthe previously identified B-cell epitope MKNNPIYSEGSL, and anothercontained a similar but not identical amino acid sequence, MKNNPVYSE-SL(Fig. 1B).

ELISA using an 11-mer synthetic peptide, MKNNPVYSESL. Because the 11-mer amino acid (G13), MKNNPVYSESL, was very similarto the previously identified 12-mer B-cell epitope (G1), weexpected that EHV-4-infected horse sera would also recognizethe peptide. However, horse sera experimentally infected withTH20p did not react with the peptide, and the reaction to G13was significantly less than that to G1 (P < 0.01) (Fig. 2).Paired sera from horses naturally infected with EHV-4 were examinedby ELISA using G1 or G13 as antigen. The results revealed thatG13 significantly reacted with EHV-4-infected horse sera (Fig.3). However, the reaction to G13 was observed to be less thanthat to G1, and paired sera from one EHV-4-infected horse didnot react with G13 at all (Fig. 3, open circle).

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FIG. 2. ELISA using two synthetic peptides against sera from horses experimentally infected with EHV-4. Two synthetic peptides (G1 and G13) were used as ELISA antigens. Sera collected from six foals that were experimentally infected with EHV-4 TH20p were diluted 1:1,000. Peptide reactivity is shown as the mean absorbance at 405 nm. Vertical error bars represent standard deviations of the means.

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FIG. 3. ELISA using G1 or G13 against sera from horses naturally infected with EHV-4. Both G1 (A) and G13 (B) were used as ELISA antigens, and a 1:125 dilution of paired sera collected during acute and convalescent phases from horses with pyrexia caused by EHV-4 infection were used. Open circles indicate the horse whose sera reacted to G1 but not to G13.

DISCUSSION

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Discussion

Heterogeneity among field isolates was found in the type-specificregion of EHV-4 gG (Fig. 1). Because field isolates were propagatedless than three times in FHK cells, the heterogeneity observedis likely a natural characteristic of the EHV-4 field isolates.Only the H45 strain had four repeat sequences. Because the H45strain was isolated in the 1960s and propagated in vitro numeroustimes, the high passage number might have influenced the numberof repeat sequences.

Comparison of amino acid sequences showed that three isolates,87c34, H45, and NS80567, had two kinds of repeat sequences.One contained a B-cell epitope (G1), and the other one had asimilar, but not identical, sequence (G13), MKNNPVYSE-SL (Fig.1B). On the other hand, TH20p had repeat sequences containingonly G1. Because G1 is a B-cell epitope (4), we speculated thatG13, which is similar to G1, could also be recognized by EHV-4-specificantibodies. However, G13 was not recognized by sera from horsesexperimentally infected with TH20p (Fig. 2), indicating thatanti-G1 antibodies are not cross-reactive with G13. On the otherhand, sera from horses naturally infected with EHV-4 reactedwith both G1 and G13. These results indicated that most EHV-4-infectedhorses have antibodies to G13 as well as to G1 and that G13is possibly another B-cell epitope in the type-specific region.It is not clear why the antibody specific to G1 did not cross-reactwith G13. It is speculated that loss of glycine and change fromisoleucine to valine might result in the loss of antibody-bindingaffinity. In addition, it is interesting that both of the twopeptides still possess immunogenicity to induce antibodies tohorses in spite of their different specificities.

Although horses naturally infected with EHV-4 had antibodiesto G13 as well as G1 (Fig. 3), TH20p-infected horses had antibodiesto G1 only (Fig. 2). These results are understandable, as TH20pdid not have a repeat sequence containing G13. Comparison ofELISA using G1 and G13 showed that most horses had antibodiesto both epitopes, except for one horse that had antibodies onlyto G1 (Fig. 3, open circles). These results indicated that mostEHV-4 probably have repeat sequences containing both G1 andG13, though some EHV-4, including TH20p, have repeat sequencescontaining only G1. To the best of our knowledge, all EHV-4field isolates had the repeat sequence containing G1, indicatingthat ELISA using G1 may be sufficient for seroepizootiologicalstudies and for the diagnosis of EHV-4. However, there is apossibility that some field isolates might have repeat sequencescontaining only G13 and not G1. Therefore, both G1 and G13 shouldbe used as ELISA antigens for the diagnosis and epizootiologicalstudies of EHV-4.

In this study, heterogeneity in the type-specific region wascompared among field isolates, and another B-cell epitope, G13,was identified. Our study indicated that ELISA using both G1and G13 as antigens should be conducted for seroepizootiologicalstudies and diagnosis of EHV-4 infection.

ACKNOWLEDGMENTS

This study was supported by a grant-in-aid from the Ministryof Education, Culture, Sports, Science and Technology of Japan.

We thank Malaika Watanabe for suggestions to improve our manuscript.

FOOTNOTES

* Department of Veterinary Microbiology, Faculty of Agriculture, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8515, Japan. Phone and fax: 81-83-933-5887. E-mail: kmaeda{at}yamaguchi-u.ac.jp.

REFERENCES

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