The equine herpesvirus type 1 (EHV-1) homolog of herpes simplex virus type 1 US9 and the nature of a major deletion within the unique short segment of the EHV-1 KyA strain genome.

The DNA sequence of the short (S) genomic component of the equine herpesvirus type 1 (EHV-1)KyA strain has been determined recently in our laboratory. Analysis of a 1353-bp BamHI/PvuII clone mapping at the unique short/terminal inverted repeat (Us/TR) junction revealed 507 bp of Us and 846 bp of TR sequences as well as an open reading frame (ORF) that is contained entirely within the Us. This ORF encodes a potential polypeptide of 219 amino acids that shows significant homology to the US9 proteins of herpes simplex virus type 1 (HSV-1), EHV-4, pseudorabies virus (PRV), and varicella zoster virus (VZV). The US9 polypeptides of the two equine herpesviruses exhibit 50% identity but are twice as large as their counterparts in HSV-1, PRV, and VZV. All five US9 proteins are enriched for serine and threonine residues and share a conserved domain of highly basic residues followed by a region of nonpolar amino acids. DNA sequence and Southern blot hybridization analyses revealed that the Us of EHV-1 KyA differs from the Us of EHV-1 KyD and AB1 in that the ORFs encoding glycoproteins I and E and a unique 10-kDa polypeptide are deleted from the KyA genome. These data demonstrate that the predicted 10-kDa protein unique to EHV-1 is nonessential for replication in vitro and that EHV-1 glycoproteins I and E, like their equivalents in HSV-1 and PRV, are also nonessential. These findings and those reported previously by this laboratory and others reveal that the Us segment of EHV-1 comprises nine ORFs, two of which, US4 and 10-kDa ORF, are unique to EHV-1. The gene order of the Us is US2, protein kinase, gG, US4, gD, gI, gE, 10 kDa, and US9.     

Open reading frames encoding a protein kinase, homolog of glycoprotein gX of pseudorabies virus, and a novel glycoprotein map within the unique short segment of equine herpesvirus type 1.

DNA sequence analysis of the unique short (Us) segment of the genome of equine herpesvirus type 1 Kentucky A strain (EHV-1) by our laboratory and strains Kentucky D and AB1 by other workers identifies a total of nine open reading frames (ORF). In this report, we present the DNA sequence of three of these newly identified ORFs, designated EUS 2, EUS 3, and EUS 4. The EUS 2 ORF is 1146 nucleotides (nt) in length and encodes a potential protein of 382 amino acids. Cis-regulatory sequences upstream of the putative ATG start codon include a G/C box 112 nt upstream and two potential TATA-like elements located between 15 and 90 nt before the ATG. The EUS 2 translation product exhibits significant homology to Ser/Thr protein kinases encoded within the Us segments of other herpesviruses, such as herpes simplex virus (26% homology) and pseudorabies virus (PRV), (45% homology), and possesses sequence domains conserved in protein kinases of cellular and viral origin. The EUS 3 ORF begins 127 nt downstream from the EUS 2 stop codon and ends at a stop codon 1119 nt further downstream. A single TATA-like element maps 61 nt upstream of the ORF. This ORF encodes a potential protein of 373 amino acids and is a homolog of glycoprotein gX of PRV, as judged by overall homology of amino acid residues, cysteine displacement, and presence of potential glycosylation sites and signal sequence. Interestingly, the EUS 4 ORF encodes a potential membrane glycoprotein that does not exhibit homology to any reported protein sequence. The EUS 4 ORF encodes a 383 amino acid polypeptide with a sequence indicative of a signal sequence at its amino terminal end, glycosylation sites for N-linked oligosaccharides, and a transmembrane domain near its carboxyl terminus. Several cis-acting regulatory sequences lie upstream of this ORF. These findings support the observation that the short region of alphaherpesviruses show considerable variation in their genetic content and gene organization.     

Contribution of gene products encoded within the unique short segment of equine herpesvirus 1 to virulence in a murine model

The pathogenesis of three equine herpesvirus 1 (EHV-1) recombinants was assessed in a CBA mouse model. Sequences encoding the majority of glycoproteins I (gI) and E (gE) were deleted from the pathogenic EHV-1 strain RacL11 (L11ΔgIΔgE), and sequences comprising the 3859 bp deletion within the strain KyA U_S segment, which includes genes 73 (gI), 74 (gE), and 75 (putative 10 kDa protein 75), were re-inserted into attenuated KyA (KgI/gE/75). In addition, genes gE and 75 were inserted into KyA to generate the EHV-1 recombinant KgE/75. The insertion of the 3859 bp U_S segment was sufficient to confer virulence to KyA, as indicated by pronounced signs of clinical disease including substantial weight loss. A large plaque morphology was observed in cells infected with KgI/gE/75 compared with KyA, and a small plaque phenotype was observed in cells infected with L11ΔgIΔgE compared with RacL11. These data indicate that gI and/or gI and gE contribute to the ability of EHV-1 to spread directly from cell-to-cell. The deletion of both gI and gE from the pathogenic RacL11 strain did not reduce clinical signs of disease in infected mice, but did decrease mortality compared with RacL11. Furthermore, the insertion of genes 74 (gE) and 75 into the vaccine strain KyA did not alter the attenuated phenotype of this virus. Finally, KgI/gE/75 and RacL11 elicited the production of the proinflammatory chemokines MIP-1, MIP-1β, and MIP-2 in the lungs of infected mice, while KyA did not, suggesting that gI and/or gI and gE contribute to the up-regulation of these mediators of inflammation. These findings show that gI, and/or gI and gE restore a virulent phenotype to the EHV-1 KyA strain, and indicate that virulence factors, in addition to gI and gE, contribute to the pathogenesis of the RacL11 strain.     

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.     

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.     

Meningoencephalitis in Mice Infected with an Equine Herpesvirus 1 Strain KyA Recombinant Expressing Glycoprotein I and Glycoprotein E

One of the consequences of equine herpesvirus 1 (EHV-1) infection in the natural host is a neurological disease that can lead to paralysis. The pathology associated with EHV-1-induced neurological disease includes vasculitis of the small blood vessels within the central nervous system and subsequent damage to the surrounding neural tissue. In a previous study, an EHV-1 recombinant KyA virus (KgI/gE/75) was generated in which the sequences encoding glycoprotein I (gI) and glycoprotein E (gE) were repaired [Frampton et al. 2002 (Virus Research 90: 287-301)] using genes of the pathogenic EHV-1 strain 89c25. In contrast to the parental KyA virus that lacks gI and gE, the recombinant KgI/gE/75 was able to spread to the brains of CBA mice after intranasal infection. Infection resulted in a meningoencephalitis characterized by lymphocytic cuffing of small blood vessels within the brain, consistent with that observed in EHV-1-infected horses exhibiting neurological signs. KgI/gE/75 was able to elicit cytopathology in the lung prior to spread to the brain. However, like the attenuated KyA strain, KgI/gE/75 did not persist in the lung and was completely cleared from lung tissue by day 5 postinfection. We propose that gI and gE are neurovirulence factors for EHV-1, and that the CBA mouse model can be extended to study neurologic sequelae resulting after EHV-1 infection.     

Use of λgt11 to identify antigenic components of equine herpesvirus 4

A library of the equine herpesvirus 4 (EHV-4) genome was constructed in the gt11 expression vector. Recombinant bacteriophage expressing EHV-4 antigens as beta-galactosidase fusion proteins were detected with rabbit antiserum raised against EHV-4 virions and convalescent horse serum. EHV-4 DNA sequences contained in the immunopositive recombinants were used as hybridization probes for mapping the genes encoding the antigens on the viral genome. The DNA sequence of the probes was determined. Screening the library with rabbit antiserum led to the identification of 40 recombinants, 26 of which were further characterized. Determination of the DNA sequence of the EHV-4 inserts revealed that 23 of the recombinants encode an identical portion of glycoprotein gB. Two of the recombinants encode a portion of the previously unidentified EHV-4 homologue of the EHV-1 immediate early protein. The EHV-4 insert of the remaining recombinant encodes a portion of the previously unidentified EHV-4 homologue of herpes simplex virus 1 (HSV-1) UL36, a tegument protein. Screening the library horse serum led to the identification of three recombinants, one of which encodes the same gB sequence as the gB recombinant recognized with the rabbit serum. The other two contain overlapping sequences that encode a portion of EHV-4 gX.     

Localization of the UsProtein Kinase of Equine Herpesvirus Type 1 Is Affected by the Cytoplasmic Structures Formed by the Novel IR6 Protein

Previous work revealed that the Us(unique short) segment of equine herpesvirus type-1 (EHV-1), like that of other alphaherpesviruses, encodes a serine/threonine protein kinase (PK). Experiments were carried out to identify the PK encoded by the EHV-1 EUS2 gene (ORF 69) and to ascertain its time course of synthesis and cellular localization. Western blot and immunoprecipitation analyses of EHV-1-infected cell extracts using a PK-specific polyclonal antibody generated against a bacterially expressed TrpE/PK fusion protein identified the UsPK as a 42- to 45-kDa phosphoprotein. The PK protein is first synthesized at 3 hr postinfection, is produced throughout the infection cycle, and is incorporated into EHV-1 virions. Interestingly, immunoprecipitation analyses revealed that the PK protein within the cytoplasm is associated with the 33-kDa IR6 novel protein of EHV-1, is expressed abundantly as an early protein, and is present in the large rod-like structures formed by the IR6 protein (ORF67 protein) within the cytoplasm of infected cells. Confocal microscopic examination of cells stained with fluorescein-labeled antibody clearly showed that the PK protein colocalized with the cytoplasmic IR6 rod-like structures and remained associated with these unique structures during infection. In contrast, in cells infected with the EHV-1 RacM strain in which the IR6 protein harbors four amino acid substitutions that prevent formation of the rod-like structures (Osterriederet al.,1996,Virology217, 442–451), the PK protein localized predominantly to the nucleus. The possible significance of the association of the IR6 and PK proteins in EHV-1 replication is discussed.     

Generation and characterization of an EICP0 null mutant of equine herpesvirus 1

The EICP0 gene (gene 63) of equine herpesvirus 1 (EHV-1) encodes an early regulatory protein that is a promiscuous trans-activator of all classes of viral genes. Bacterial artificial chromosome (BAC) technology and RecE/T cloning were employed to delete the EICP0 gene from EHV-1 strain KyA. Polymerase chain reaction, Southern blot analysis, and DNA sequencing confirmed the deletion of the EICP0 gene and its replacement with a kanamycin resistance gene in mutant KyA. Transfection of rabbit kidney cells with the EICP0 mutant genome produced infectious virus, indicating that the EICP0 gene is not essential for KyA replication in cell culture. Experiments to assess the effect of the EICP0 deletion on EHV-1 gene programming revealed that mRNA expression of the immediate-early gene and representative early and late genes as well as the synthesis of these viral proteins were reduced as compared to the kinetics of viral mRNA and protein synthesis observed for the wild type virus. However, the transition from early to late viral gene expression was not prevented or delayed, suggesting that the absence of the EICP0 gene did not disrupt the temporal aspects of EHV-1 gene regulation. The extracellular virus titer and plaque areas of the EICP0 mutant virus KyADeltaEICP0, in which the gp2-encoding gene 71 gene that is absent in the KyA BAC was restored, were reduced by 10-fold and 19%, respectively, when compared to parental KyA virus; while the titer and plaque areas of mutant KyADeltaEICP0Deltagp2 that lacks both the EICP0 gene and gene 71 were reduced more than 50-fold and 67%, respectively. The above results show that the EICP0 gene is dispensable for EHV-1 replication in cell culture, and that the switch from early to late viral gene expression for the representative genes examined does not require the EICP0 protein, but that the EICP0 protein may be structurally required for virus egress and cell-to-cell spread.