Analysis of equine herpesvirus 2 strain variation using monoclonal antibodies to glycoprotein B

Summary.  The antigenic relationships of four genomically divergent strains of equine herpesvirus 2 (EHV2.86/67, EHV2.5FN, EHV2.141 and EHV2.T-2) and equine herpesvirus 5 (EHV5) were examined in ELISA using a panel of EHV2.86/67 gB-specific MAbs. EHV2.86/67 and EHV2.5FN were shown to be more similar to each other than to EHV2.T-2, EHV2.141 or EHV5. Seven of nine EHV2.86/67 gB specific MAbs tested in serum neutralisation assays were shown to neutralise EHV2.86/67 and EHV2.5FN but not EHV2.141, EHV2.T-2 or EHV5. The complete nucleotide and deduced amino acid sequences of EHV2.86/67, EHV2.5FN, EHV2.141 and EHV2.T-2 gB were compared and contrasted with each other and with EHV5 gB. The four EHV2 strains were 94–96% similar at the amino acid level and variability in amino acid sequence mapped to three mains sites designated I, II and III. By contrast, the four EHV2 strains were 77–79% similar to EHV5 gB at the amino acid level. The epitope of these seven gB specific neutralising MAbs has been previously mapped to amino acids 29–74 of EHV2 gB and examination of the deduced amino acid sequence of the four sequenced strains localised the epitope of the seven MAbs to amino acids 30 to 49 located within Site I. Six other divergent strains of EHV2 were examined for variability at Site I using DNA sequencing. Examination of the deduced amino acid sequences of all ten EHV2 strains tested indicated, that based on the epitope of the neutralising MAbs the EHV2 strains formed two distinct antigenic groups, EHV2.86/67-like and EHV2.141-like. EHV5 gB showed divergence from all of the EHV2 gB sequences between amino acids 29–74. Received July 19, 1999 Accepted January 20, 2000     

Evidence for involvement of equine herpesvirus 1 glycoprotein B in cell-cell fusion

Summary Monoclonal antibodies specific for equine herpesvirus 1 (EHV-1) glycoproteins (gB, gD, gp2 and a cleaved translation product of gene 71) were tested for ability to inhibit cell-cell fusion as measured by syncytium formation in EHV-1 infected cell cultures. Syncytium formation was inhibited by a complement-dependent neutralising antibody (7B10) which recognised the large subunit of EHV-1 gB. This indicated that EHV-1 gB, in common with gB homologues of herpes simplex virus and other herpesviruses, plays a role in the cell-cell fusion process.     

Construction and characterization of an equine herpesvirus 1 glycoprotein C negative mutant

An equine herpesvirus 1 (EHV-1) strain RacL 11 mutant was constructed that carries the Escherichia coli LacZ gene instead of the open reading frame encoding glycoprotein C (gC). The engineered virus mutant (L11(delta)gC) lacked codons 46-440 of the 1404 bp gene. On rabbit kidney cell line Rk13 and equine dermal cell line Edmin337, the L11(delta)gC virus grew to titers which were reduced by approximately 5- to 10-fold compared with wild-type RacL11 virus or a repaired virus (R-L11(delta)gC). However, when L11(delta)gC growth properties were analyzed on primary equine cells a decrease of viral titers was observed such that extracellular L11(delta)gC titers were reduced by 48- to 210-fold compared with those of wild-type or repaired virus. Heparin sensitive and heparin resistant attachment was assessed by binding studies using radiolabeled virion preparations. These studies revealed that EHV-1 gC is important for heparin sensitive attachment to the target cell. Similar results were obtained when cellular glycosaminoglycan (GAG) synthesis was inhibited by chlorate treatment or when cells defective in GAG synthesis were used. L11(delta)gC also exhibited significantly delayed penetration kinetics on Rk13 and primary equine cells. Infection of mice with L11(delta)gC did not cause EHV-1-related disease, whereas mice infected with either RacL11 or R-L11(delta)gC exhibited massive bodyweight losses, high virus titers in the lungs, and viremia. Taken together, EHV-1 gC was shown to play important roles in the early steps of infection and in release of virions, especially in primary equine cells, and contributes to EHV-1 virulence.     

Identification of equine herpesvirus 4 glycoprotein G: a type-specific, secreted glycoprotein.

Equine herpesvirus 4 (EHV4) glycoproteins of M(r) 63K and 250K were identified in the supernatant of infected cell cultures. The 63K glycoprotein was type-specific; that is, it reacted with monospecific sera from horses that had been immunized or infected with EHV4, but not with monospecific sera from horses immunized or infected with EHV1, a closely related alphaherpesvirus. It was postulated that the secreted protein may be the homologue of similarly secreted glycoproteins of herpes simplex virus 2 glycoprotein G (HSV2 gG) and pseudorabies virus (PRV) gX, which is the homologue of HSV2 gG. The US region of the EHV4 genome, toward the internal repeat structure, was sequenced. Four open reading frames (ORFs) were identified of which ORF4 showed 52% similarity to the gene-encoding PRV gX in a 650-nucleotide region. ORF4 coded for a primary translational product of 405 amino acids which has a predicted size of 44K. The amino acid sequence of ORF4 showed 28% identity with PRV gX and 16% identity with HSV2 gG, although significantly greater identity was observed in the N-terminal region including the conservation of 4 cysteine residues. Accordingly, we designate ORF4 as EHV4 gG. The predicted amino acid sequence of the EHV4 gG showed characteristics of an envelope glycoprotein. Expression of the entire EHV4 gG gene in the bacterial expression vector pGEX-3X produced a type-specific fusion protein of M(r) 70K of which the gG portion composes 43K. Antibody that was affinity purified from selected portions of Western blots containing the 70K gG fusion protein reacted with the 63K secreted glycoprotein. Conversely, antibody affinity purified to the 63K secreted product reacted with the 70K gG fusion protein. These results showed that the EHV4 63K secreted glycoprotein was EHV4 gG, the third alphaherpesvirus gG homologue known to be, at least in part, secreted. The type-specificity of this glycoprotein provides, for the first time, the opportunity to differentiate between antibodies present in polyclonal sera from EHV4, EHV1, and dual-infected horses and this has important implications for understanding the epidemiology of these viruses.     

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 the type-specific region of equine herpesvirus 1 (EHV-1) glycoprotein G (gG) was identified and used as an antigen for enzyme-linked immunosorbent assay (Maeda et al., J. Clin. Microbiol. 42:1095-1098, 2004). Although our prototype strain, TH20p, possesses two repeat sequences containing the B-cell epitope, the EHV-4 NS80567 strain has two repeat sequences that are not identical. One repeat sequence stretch contained the B-cell epitope, while the other contained the 11-mer, MKNNPVYSESL (underlining indicates a different amino acid). In this study, heterogeneity of the type-specific region was compared among Japanese EHV-4 isolates. The 11-mer peptide, MKNNPVYSESL, specifically reacted with sera from horses naturally infected with EHV-4 but not with sera from horses experimentally infected with EHV-4 TH20p. The 11-mer peptide may be another B-cell epitope in the type-specific region.     

Rapid subtyping of equine herpesvirus 1 with monoclonal antibodies.

Two antigenically similar subtypes of equine herpesvirus 1 (EHV-1) cause disease in horses. A procedure for rapid differentiation of the two EHV-1 subtypes with monoclonal antibodies was developed. Subtype-specific pools of monoclonal antibodies were constructed, characterized, and used in enzyme immunofiltration and indirect immunofluorescence assays to subtype 50 epizootiologically unrelated field isolates of EHV-1. Both assays allowed accurate subtype identification of each EHV-1 isolate with the monoclonal antibody pools. The subtyping procedures were simple and amenable to typing many isolates at one time and permitted unambiguous EHV-1 subtype identification with 3 h after isolation of the virus in tissue culture.     

Synthesis and processing of equine herpesvirus type 1 glycoprotein 14

Glycoprotein 14 (gp14) of equine herpesvirus type 1 (EHV-1), the homolog of herpes simplex virus (HSV) glycoprotein B (gB), was investigated employing a panel of monoclonal antibodies to ascertain the regulatory class, rate of synthesis, and type of glycosylation of this polypeptide. Application of immunoprecipitation, Western blot, and SDS-PAGE analysis in conjunction with the use of metabolic inhibitors (cycloheximide, antinomycin D, phosphonoacetic acid, tunicamycin, and monensin), and time-course and pulse-chase experiments revealed the following information: (1) Three gp14-related polypeptides with molecular weights of 138 kilodaltons (K), 77-75K, and 55-53K are present in EHV-1-infected cell extracts. (2) All three species are synthesized in the presence of the DNA synthesis inhibitor phosphonoacetic acid although their synthesis is enhanced by DNA replication, indicative of a beta-gamma class molecule. (3) The 138K species is synthesized first as a precursor of the smaller species of gp14, the 77-75K and 55-53K forms. (4) Use of glycosylation inhibitors and digestion of immunoprecipitated gp14 with endoglycosidases indicate that the primary translation product is a 118K molecule which is cotranslationally glycosylated to the 138K form by the addition of high mannose oligosaccharides. (5) The 77-75K species contains both high mannose and hybrid oligosaccharides while the 55-53K form of gp14 contains some complex oligosaccharides. (6) In the absence of a reducing agent, the 138K polypeptide and a large 145K species are observed in both infected cell extracts and purified virions. Thus, EHV-1 gp14 appears to be synthesized as a large precursor molecule of 138K and is proteolytically cleaved to two smaller forms, 77-75K and 55-53K, which are linked by a disulfide bond(s) to form a 145K complex. This model of gp14 synthesis and maturation is similar to those proposed for a number of HSV gB equivalents found in the Alphaherpesvirnae.     

Identification, sequence analysis and characterisation of equine herpesvirus 5 glycoprotein B

Summary.  The complete nucleotide sequence of the gammaherpesvirus equine herpesvirus 5 (EHV5) glycoprotein B (gB) was determined and the deduced amino acid sequence compared with that of the second equine gammaherpesvirus EHV2. EHV5 gB is an 870 amino acid protein and is 79% similar and 66% identical with EHV2 gB at the amino acid level. EHV5 gB like EHV2 gB is a disulphide linked heterodimer with subunits of 92 and 68 kDa. EHV5 gB is an integral membrane glycoprotein containing only N-linked oligosaccharides and contains a putative endoproteolytic cleavages site at amino acids 422–485. The EHV5 gB amino acid sequence showed greatest homology with other members of the Rhadinovirus genus of the subfamily Gammaherpesvirinae. Alignment of EHV5 gB sequence with the gB sequence of seven other gammaherpesviruses showed conservation of 10 cysteine residues as well as conservation of three predicted sites of N-linked glycosylation; the highest degree of conservation of the predicted sites of N-linked glycosylation was observed between EHV5 and the other members of the Rhadinovirus genus. Phylogenetic analysis confirmed EHV2 and EHV5 were most closely related to each other and equally distant from other members of the Rhadinovirus genus included in the analysis. Accepted September 7, 1998 Received March 5, 1998     

Detection of equine herpesvirus type 1 by real time PCR

A real-time PCR assay was developed for detection and quantitation of equid herpesvirus type 1 (EHV-1). The sensitivity of the assay was compared with an established nested-PCR (n-PCR). The real-time PCR detected 1 copy of target DNA, with a sensitivity 1 log higher than gel-based n-PCR. The assay was able to detect specifically EHV-1 DNA in equine tissue samples and there was no cross-amplification of other horse herpesviruses. Real-time PCR was applied to determine EHV-1 load in tissue samples from equine aborted fetuses. The high sensitivity and reproducibility of the EHV-1-specific fluorogenic PCR assay, combined with the wide dynamic range and the high throughput, make this method suitable for diagnostic and research applications.     

Detection of equine herpesvirus type 1 using a real-time polymerase chain reaction

Equid herpesvirus 1 (EHV1) is a major disease of equids worldwide causing considerable losses to the horse industry. A variety of techniques, including PCR have been used to diagnose EHV1. Some of these PCRs were used in combination with other techniques such as restriction enzyme analysis (REA) or hybridisation, making them cumbersome for routine diagnostic testing and increasing the chances of cross-contamination. Furthermore, they involve the use of suspected carcinogens such as ethidium bromide and ultraviolet light. In this paper, we describe a real-time PCR, which uses minor groove-binding probe (MGB) technology for the diagnosis of EHV1. This technique does not require post-PCR manipulations thereby reducing the risk of cross-contamination. Most importantly, the technique is specific; it was able to differentiate EHV1 from the closely related member of the Alphaherpesvirinae, equid herpesvirus 4 (EHV4). It was not reactive with common opportunistic pathogens such as Escherichia coli, Klebsiella oxytoca, Pseudomonas aeruginosa and Enterobacter agglomerans often involved in abortion. Similarly, it did not react with equine pathogens such as Streptococcus equi, Streptococcus equisimilis, Streptococcus zooepidemicus, Taylorella equigenitalis and Rhodococcus equi, which also cause abortion. The results obtained with this technique agreed with results from published PCR methods. The assay was sensitive enough to detect EHV1 sequences in paraffin-embedded tissues and clinical samples. When compared to virus isolation, the test was more sensitive. This test will be useful for the routine diagnosis of EHV1 based on its specificity, sensitivity, ease of performance and rapidity.     

Detection of bovine herpesvirus 4 glycoprotein B and thymidine kinase DNA by PCR assays in bovine milk

A polymerase chain reaction (PCR) assay was developed to detect bovine herpesvirus 4 (BHV4) glycoprotein B (gB) DNA, and a nested-PCR assay was modified for the detection of BHV4 thymidine kinase (TK) DNA in bovine milk samples. To identify false-negative PCR results, internal control templates were constructed, added to milk samples, and co-amplified with viral DNA using the same primers for both templates. Specificity, sensitivity, and reproducibility of the two PCR assays were examined. In both PCR assays, all 31 BHV4 strains examined were scored positive, whereas 14 unrelated viruses scored negative. Sensitivity studies showed that two-ten copies of BHV4 DNA were detectable by the gB-PCR, while one-three copies could be detected by the TK-PCR. For the detection of BHV4 in milk samples, the gB-PCR amplification was found to be ten-times, and the TK-PCR was found to be 55-times more sensitive than virus isolation. BHV4 DNA was detected by gB-PCR and TK-PCR in 93 and 95%, respectively, of 61 milk samples collected from cows infected intramammarily with BHV4, while only 61% were positive by virus isolation. Four out of 48 cows with clinical mastitis were positive for BHV4-gB and BHV4-TK DNA, whereas no BHV4 DNA was detected in milk from control cows. Considerable agreement was seen between the results of the two PCR assays, and both methods were considered as rapid and reliable tests for the screening of BHV4 DNA in bovine milk. The less laborious gB-PCR might be the recommended test of choice for screening large amounts of milk samples for the presence of BHV4.     

Binding behaviour of antibodies reacting specifically with an immunodominant region of the transmembrane protein gp41 of HIV-1.

A highly conserved region of the transmembrane protein gp41 of the HIV-1 was found in which the amino acid sequence 606 to 620 was characterized as an immunodominant region. The binding behaviours of a human monoclonal antibody and of polyclonal specific antibodies in sera of HIV-infected persons to this particular sequence have been studied. In two sandwich-type ELISAs with synthetic peptides as antigen representing this region, sera of 312 HIV-infected persons at all stages could be clearly discriminated against 500 anti-HIV antibody negative sera of healthy blood donors.     

Monoclonal antibodies directed against a 130K glycoprotein of bovine herpesvirus 2 cross-react with glycoprotein B of herpes simplex virus

Envelope proteins of bovine herpesvirus type 2 (BHV-2) and herpes simplex virus types 1 and 2 (HSV-1, -2) share cross-reacting determinants. Monoclonal antibodies directed against epitopes of a 130K glycoprotein of BHV-2 detect determinants on the surface of infected cells and react either with cytoplasmic or nuclear antigens. Biological and biochemical characterization of the proteins recognized by these antibodies revealed that the BHV-2 specific 130K glycoprotein and the gB of HSV share common epitopes. Some of them are involved in the neutralization of HSV and BHV-2.     

Cytokine profiles and long-term virus-specific antibodies following immunization of CBA mice with equine herpesvirus 1 and viral glycoprotein D

Equine herpesvirus 1 (EHV-1)-specific antibody-secreting cells (ASC) isolated from the lung and spleen of mice at 12 months after immunization with attenuated EHV-1 KyA, heat-killed KyA, or recombinant viral glycoprotein D (rgD) assessed by ELISPOT showed a three- to fivefold increase in three immunoglobulin isotypes at 3 days post-challenge with pathogenic EHV-1 RacL11 as compared to control mice. ELISPOT assays demonstrated a high frequency of cells secreting proinflammatory tumor necrosis factor-alpha (TNF-alpha), interferon gamma (IFN-gamma), and interleukin 4 (IL-4) in the lungs in response to infection with KyA or RacL11 or immunization with rgD. Cytokine production elicited by EHV-1 KyA or RacL11 infection revealed similar frequencies of EHV-1-specific IFN-gamma and IL-4 spot forming cells in the mediastinal lymph nodes and spleen. However, KyA induced significantly greater amounts of IFN-gamma producing cells in the lungs than did RacL11. Intranasal immunization with KyA or rgD induced long-term immunity that provided protection against pathogenic EHV-1 challenge infection at 12 months post-immunization. Overall, the data indicate that immunization with infectious KyA or rgD induces significant levels of cytokines, virus-specific ASC in the lungs and spleen, and long-term virus specific B-cell responses.     

In vitro neutralization of equid herpesvirus 1 mediated by recombinant antibodies

A phage antibody display library of single chain fragment variables (scFv) was applied to develop anti-equid herpesvirus-1 (EHV-1) glycoprotein D (gD) neutralizing antibodies. To enrich for specific scFvs, the phage antibody library was panned against epitope derived from the N-terminal part of EHV-1 gD. Unique clones were differentiated by BstNI fingerprinting and further characterized by sequencing and immunoreactivity. The neutralizing effect of each clone was assessed by plaque reduction assay. Three clones with neutralizing effect were isolated.     

Quantitation of virus-specific classes of antibodies following immunization of mice with attenuated equine herpesvirus 1 and viral glycoprotein D

The antibody responses of CBA/J mice infected intranasally (i.n.) with either the attenuated KyA strain or the pathogenic RacL11 strain of equine herpesvirus 1 (EHV-1) or immunized with recombinant glycoprotein D (rgD) were investigated using the ELISPOT assay to measure EHV-1-specific antibody-secreting cells (ASC) in the regional lymphoid tissue of the respiratory tract. IgG, IgA, and IgM ASC specific for EHV-1 were detected in the mediastinal lymph nodes (MLN) and lungs 2 weeks after i.n. infection with EHV-1 strain KyA or RacL11, or immunization with heat-killed KyA or rgD. EHV-1-specific ASC were present in the MLN and lungs at 4 and 8 weeks, but declined in frequency by fivefold in the lung at 8 weeks. However, i.n. immunized (2 x 10(6) pfu KyA or 50 microgram rgD/mouse) mice infected at 8 weeks with pathogenic EHV-1 RacL11 resisted challenge and showed eight- and tenfold increases in MLN ASC and lung ASC, respectively, by 3 days after challenge. In contrast to the intranasal route of immunization, intraperitoneal immunization yielded ASC frequencies in the MLN and lungs that were only slightly above those of nonimmunized control mice. These data indicate that immunization with infectious or heat-killed EHV-1 KyA, or rgD, induces significant levels of virus-specific ASC both in the MLN and lungs, a specific memory B-cell response, and long-term protective immunity. The finding that the numbers of ASC induced by the pathogenic strain versus the attenuated strain of EHV-1, which were virtually identical, indicated that the ability to generate a B-cell response is independent of and does not contribute to EHV-1 virulence.     

Detection of antibodies against human metapneumovirus by Western blot using recombinant nucleocapsid and matrix proteins

Detection of antibodies against individual proteins of human metapneumovirus (hMPV) is important in the analysis of immune responses to hMPV. Specific antibodies against nucleocapsid (N) and matrix (M) proteins in 97 serum samples were tested by Western blot using recombinant N and M proteins of hMPV expressed in Escherichia coli. The results were compared with those of immunofluorescence assays (IFAs) based on hMPV-infected LLC-MK2 cells, which expressed the whole hMPV proteins. Thirty (61.2%) and 31 (63.3%) of 49 serum samples with titers of > or = 1:160 by IFA reacted with N and M proteins, respectively. Only 2 (4.2%) of 11 serum samples with titers of 1:80 by IFA reacted with N and M proteins. Antibodies against N and M proteins were not detected in 37 serum samples with titers of < 1:40 by IFA. These results indicate that the antibodies against N and M proteins are highly specific (100%) but less sensitive (42.1%, N protein; 40.8%, M protein) than those against whole proteins of hMPV detected by IFA. The reactivity of sera with the recombinant N protein and that with the recombinant M protein correlated well (correlation coefficient of 0.79), and the concordance of reactivities was 91% (kappa = 0.79). In summary, both recombinant N and M proteins of hMPV were antigenic, and the responses to N and M protein varied among patients. Therefore, Western blot using N and M proteins provide a useful tool for analysis of immune responses to hMPV.     

Measurement of antibodies to herpesvirus types 1 and 2 in human sera by microradioimmunoassay.

The binding of 125I-labelled anti-human antibodies against the fc IgG fragment to unlabelled antiviral immunoglobulins in the surface of infected cells was used to quantitate antibodies against herpes simplex virus type (HSV-1) and type 2 (HSV-2) in sera from patients with cervix carcinoma. The microradioimmunoassay technique (micro-RIA) proved to be 5-10 times more sensitive than the microneutralization test. Antibody titres determined by micro-RIA correlated with neutralizing antibody titres to both HSV-1 and HSV-2. The relative antibody titres to HSV-1 and HSV-2, as determined by micro-RIA, could be used to distinguish persons previously infected with HSV-2 by means of II/I indices.