Equine herpesvirus 1 (EHV-1), a member of Varicellovirus genus in the Alphaherpesvirinae subfamily, is one of the most prevalent viruses affecting equine populations worldwide. Infection with EHV-1 may result in rhinopneumonitis, abortion, and often fatal encephalomyelitis. The continuing threat imposed by EHV-1 results in ongoing efforts aiming at a better understanding of EHV-1 pathogenesis and at developing safe and effective vaccines (27). Herpesviral glycoproteins are involved in different stages of virus infection and are major targets of the host's immune response. Envelope glycoproteins of EHV-1 are designated gB, gC, gD, gE, gG, gH, gI, gK, gL, and gM according their homologues in herpes simplex virus type 1 (HSV-1) (reviewed in reference 28). Compared to HSV-1 and other Alphaherpesvirinae, however, EHV-1 encodes two additional glycoproteins. One of the glycoproteins unique to EHV-1 was originally designated gp2 or gp300 (2, 45) and is expressed from gene 71 (EUS4) (38,42). Homologues of gp2 are found in EHV-1, EHV-4, and asinine herpesvirus 3 (8) and are among the mostabundant and -immunogenic glycoproteins in EHV-1 and EHV-4 virions (1, 7). EHV-1 gp2 is rich in serine and threonine residues and is a heavily O-glycosylated protein with a molecular mass in the range of 192 to Ͼ400 kDa (38,39,45). In contrast to EHV-4 gp2, the EHV-1 glycoprotein was shown to be partially cleaved into two polypeptides in infected cells (19). Endoproteolytic cleavage occurs after each of two adjacent arginine (R) residues at positions 506 and 507 in the sequence HRGRAGGR 506 R 507 G, and results in a 42-kDa carboxy-terminal subunit, which contains the transmembrane anchor, and an N-terminal serine/threonine-rich component that is highly O-glycosylated (19, 43). In the absence of gp2, EHV-1 strains were shown to be impaired in virus egress, while secondary envelopment appears to occur with unaltered kinetics and efficiency (31,32,37). It was also reported that deletion of gene 71 in EHV-1 strain Ab4 resulted in attenuation, and the generated virus mutant used as an experimental vaccine conferred protection against pulmonary disease in mice after challenge with wild-type virus (21).A number of EHV-1 strains with different biological properties have been described. While the RacL11 strain was isolated in the late 1950s from an aborted foal and exhibits high virulence in the natural host and laboratory animals, strain KyA, a candidate vaccine strain, is completely apathogenic for both mice and horses after serial passage in mouse L-M cells (6,25,26). As a result of a long history of passages in nonhomologous cells, KyA exhibits several genomic alterations,

Insertional mutagenesis was used to construct an equine herpesvirus 1 (EHV-1) mutant in which the open reading frame for glycoprotein D was replaced by a lacZ cassette. This gD deletion mutant (delta gD EHV-1) was unable to infect normally permissive RK cells in culture, but could be propagated in an EHV-1 gD-expressing cell line (RK/gD). Phenotypically complemented delta gD EHV-1 was able to infect RK cells, but did not spread to form syncytial plaques as seen with wild type EHV-1 or with delta gD EHV-1 infection of RK/gD cell cultures. Therefore EHV-1 gD is required for virus entry and for cell-cell fusion. The phenotypically complemented delta gD EHV-1 had very low pathogenicity in a mouse model of EHV-1 respiratory disease, compared to a fully replication-competent EHV-1 reporter virus (lacZ62/63 EHV-1). Intranasal or intramuscular inoculation of mice with delta gD EHV-1 induced protective immune responses that were similar to those elicited in mice inoculated with lacZ62/63 EHV-1 and greater than those following inoculation with UV-inactivated virus.

Signal cleavage sites of equine herpesvirus 1 (EHV-1) glycoproteins D and B (gD and gB) and an endoproteolytic cleavage site of EHV-1 gB were determined by N-terminal amino acid sequencing and compared with known cleavage sites of homologues in other herpesviruses. Signal cleavage of EHV-1 gD occurred between Arg 35 and Ala 36 in a region of basic amino acids resembling the endoproteolytic cleavage sites of viral glycoproteins, nine amino acids downstream of the predicted site, while EHV-1 gB was cleaved as predicted between Ala s5 and Val s~. Endoproteolytic cleavage of EHV-1 gB occurred between Arg 54s and Ala 5aa, 28 amino acids downstream of the cleavage site predicted from conserved sequences of other herpesvirus gB homologues. One interpretation of these data is that EHV-1 gB is cleaved internally at both sites, a possibility which was supported by the apparent molecular masses of the unglycosylated gB subunits produced in the presence of tunicamycin. This double cleavage would release a stretch of amino acids which is not present in sequenced gB molecules of other herpesviruses. Experiments with glycosylation inhibitors indicated that cleavage of EHV-1 gB can occur in the absence of glycosylation. N-terminal sequencing also determined that a 42 kDa EHV-1 glycoprotein was a product of internal cleavage of the protein encoded by gene 71. Staggered endoproteolytic cleavage after adjacent arginine residues 506 and 507 separates the 42 kDa Cterminal subunit containing all the cysteine residues from the serine and threonine rich N-terminal region.

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Equine herpesviruses 1 and 4 (EHV-1 and EHV-4) cause equine respiratory disease worldwide. However, only EHV-1 is a cause of abortion and neurological disease, despite the two viruses having all 76 genes in common. In addition EHV-1 has a broader host range in cell culture than EHV-4, as exemplified by the rabbit kidney (RK) cell line that is permissive for EHV-1, but not for EHV-4. Here we describe that when EHV-4 produced in equine cells was inoculated onto RK cells expressing glycoprotein D of EHV-1 (RKgD1), infection developed as clusters of rounded cells, and this infectivity could be passaged in RKgD1 cells. The progeny virus could also infect single RK cells, consistent with EHV-4 acquiring EHV1 gD from the complementing cell line. No such infection was observed for EHV-4 in RK cells expressing EHV-1 glycoprotein C. The results are consistent with gD homologues being major determinants of host cell tropism and raise the possibility that gD may be a factor in the differential pathogenicity of EHV-1 and EHV-4.

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