Equine Herpesvirus Type 1 Strain Research Articles

The Equine Herpesvirus 1 UL34 Gene Product Is Involved in an Early Step in Virus Egress and Can Be Efficiently Replaced by a UL34-GFP Fusion Protein

The structure and function of the equine herpesvirus type 1 (EHV-1) UL34 homologous protein were characterized. A UL34 protein-specific antiserum reacted with an M r28,000 protein that could not be detected in purified extracellular virions. Confocal laser scanning microscopy demonstrated that UL34 reactivity mainly concentrated at the nuclear rim, which changed into a punctuate and filamentous pattern at late times after infection. These changes in UL34 distribution were especially prominent when analyzing the distribution of a GFP-UL34 fusion protein. A UL34-negative EHV-1 was generated by mutagenesis of a recently established BAC clone of EHV-1 strain RacH (pRacH). Release of extracellular infectious virus was severely impaired after infection of Rk13 cells with HΔ34. Electron microscopy revealed a virtual absence of virus particles in the cytoplasm of infected cells, whereas nucleocapsid formation and maturation within the nucleus appeared unaffected. A UL34- GFP fusion protein with GFP linked to the C-terminus of UL34 was able to complement for the UL34 deletion in trans, while a GFP-UL34-fusion protein with GFP linked to the N-terminus of UL34 was able to only partially restore virus growth. It was concluded that the EHV-1 UL34 product is essential for an early step in virus egress, i.e., release of capsids from infected-cell nuclei.

Equine Herpesvirus Type 1 Devoid of gM and gp2 Is Severely Impaired in Virus Egress but Not Direct Cell-to-Cell Spread

Experiments were conducted to analyze the effects of a simultaneous deletion of glycoprotein M (gM) and glycoprotein 2 (gp2) of equine herpesvirus type 1 (EHV-1). EHV-1 strain RacH was cloned as a bacterial artificial chromosome (pRacH) by homologous recombination of a mini F plasmid into the unique short region of the genome, thereby deleting gene 71 encoding gp2. Upon transfection of the pRacH DNA into rabbit kidney RK13 cells, virus plaques were visible from day 1 after transfection. The mutant RacH virus (HΔgp2) reconstituted from pRacH lacked gene 71 and did not express gp2 as assayed by indirect immunofluorescence analysis using gp2-specific monoclonal antibodies. The HΔgp2 virus exhibited 10-fold reduced extracellular titers and an approximately 10% reduction in mean plaque diameters when compared to parental or gp2-revertant virus. The gM open reading frame was deleted from pRacH by recE/T mediated mutagenesis in Escherichia coli. The gM–gp2 double negative virus mutant (HΔgp2gM) did not express either of the deleted glycoproteins as demonstrated by indirect immunofluorescence analysis. The HΔgp2gM virus exhibited a 200-fold reduction of end-point extracellular titers when compared to parental RacH virus, which could not be compensated for by growth of the mutant virus on gM-expressing cells. After restoration of the gM open reading frame, however, growth of the mutant virus was comparable to the HΔgp2 virus. Plaque diameters of the gM–gp2 double-negative mutant were reduced by only 16% when compared to that of parental RacH virus. From the results it was concluded that the simultaneous absence of gM and gp2 had an additive effect on egress but not secondary envelopment or cell-to-cell spread of EHV-1.

The Equine Herpesvirus 1 UL45 Homolog Encodes a Glycosylated Type II Transmembrane Protein and Is Involved in Virus Egress

Experiments to analyze the product of the equine herpesvirus type 1 (EHV-1) UL45 homolog were conducted. Using an antiserum generated against the carboxylterminal 114 amino acids of the EHV-1 UL45 protein, proteins of Mr 32,000, 40,000, and 43,000 were detected specifically in EHV-1-infected cells. Neither form of the protein was located in purified virions of EHV-1 wild-type strain RacL22 or the modified live vaccine strain RacH, but UL45 was demonstrated to be expressed as a late (γ−2) protein. Fractionation of infected cells and deglycosylation experiments demonstrated that the EHV-1 UL45 protein represents a type II membrane glycoprotein. Deletion of the UL45 gene in RacL22 and RacH (LΔ45 and HΔ45) showed that UL45 is nonessential for EHV-1 growth in vitro, but that deletion reduced the viruses' replication efficiency. A marked reduction of virus release was observed although no significant influence was noticed either on plaque size or on the syncytial phenotype of the EHV-1 strain RacH.

Equine herpesvirus-1 strain KyA, a candidate vaccine strain, reduces viral titers in mice challenged with a pathogenic strain, RacL

The equine herpesvirus type-1 (EHV-1) strain Kentucky A (KyA) has a long history of repeated passage either in vivo in the Syrian hamster or in vitro in mouse L-M fibroblast tissue culture. This repeated passage in cells other than those of the natural host has caused genomic alterations of the KyA chromosome resulting in deletion of several genes or portions of open reading frames (ORFs). This report presents in vivo data from a mouse model of EHV-1 infection demonstrating the attenuated nature of EHV-1 strain KyA and that intranasal infection with KyA protects animals from subsequent challenge with a pathogenic strain, RacL, by reducing RacL viral titers in the lungs of the challenged animals. Mice infected with KyA exhibit no clinical manifestations of EHV-1 disease and do not experience the wasting that occurs with RacL infection. KyA-infected mice clear virus from the lung by day 5 post-infection (p.i.), whereas RacL infected mice have substantial virus titers (5 × 10 5 pfu/lung) at this time point. Intranasal infection with KyA followed by a challenge with RacL 4 weeks post-KyA infection results in a significant ( P = 0.0079) reduction in the lung titers of the RacL virus. RacL was identified as the virus present in the lungs of the challenged mice by a PCR assay employing primers to amplify the EUS4 gene which differs in size by 1.2 kilobase pairs (kbp) in the two strains. Importantly, the protection afforded by KyA is long lasting in that challenge with RacL 15 months after KyA infection, results in reduced virus titers and viral clearance by day 5 post-challenge. These results support the further consideration of EHV-1 KyA as a live virus vaccine.

Identification and characterization of the protein product of gene 67 in equine herpesvirus type 1 strain Ab4

Equine herpesvirus type 1 (EHV-1) strain Ab4 gene 67 has no counterpart in any herpesvirus sequenced to date. To identify and characterize the product of EHV-1 gene 67, we have expressed the putative amino acids 11 to 260 encoded by gene 67 as a beta-galactosidase fusion protein in Escherichia coli. The expressed fusion protein has been used to generate an antiserum raised against the gene 67 product. Immunoblotting and immunoprecipitation experiments have revealed that the anti-67 serum specifically recognizes a polypeptide with an M(r) of 36,000 (the 36K polypeptide) in infected cell extracts. The gene 67 protein is regulated as an early polypeptide in EHV-1 strain Ab4 infected cells and post-translational modification experiments have revealed that the protein is phosphorylated, but not glycosylated. The gene 67 protein has been transiently expressed in BHK-21/C13 cells using plasmid pCMV67, which contains the putative gene 67 ORF under the control of the cytomegalovirus immediate early promoter. Immunoblotting experiments with anti-67 have shown that the 36K protein is expressed at high levels in transfected cells. From both immunofluorescence and cellular fractionation experiments it is concluded that the gene 67 protein is associated with intracellular membranes and produces novel ribbon or filament-like structures within the cytoplasm of infected cells. We have demonstrated that the gene 67 product is a component of the virion nucleocapsid/tegument.

Transcriptional analyses of the unique short segment of EHV-1 strain Kentucky A

The unique short (Us) segment of the genome of equine herpesvirus type 1 (EHV-1) strain KyA is comprised of six open reading frames (ORFs) that encode: a) a homolog of the Us2 protein of herpes simplex virus type 1 (HSV-1); b) a serine threonine protein kinase that is a homolog of the HSV-1 Us3 protein; c) a homolog of pseudorabies virus glycoprotein gX and HSV-2 gG; d) a novel glycoprotein, EUS4, not encoded by other herpesviruses sequenced to date; e) a homolog of HSV-1 gD; and f) a homolog of HSV-1 Us9. The KyA strain is a deletion mutant that lacks Us sequences encoding gI, gE, and a potential 10 kD polypeptide, and thus may be useful as a parent virus for the generation of live virus vaccines. To complete the elucidation of the transcriptional program of the Us segment, Northern blot hybridization and S1 nuclease analyses were performed on poly(A)(+)-selected RNA isolated from infected cells maintained under early (phosphonoacetic acid-block) and late conditions. The findings revealed that the gene (EUS2 ORF) encoding the protein kinase is expressed as an early 2.9 kb transcript that overlaps and is 3' coterminal with a 1.6 kb early transcript that encodes the gG/gX homolog (EUS3 ORF). Two transcripts of 1.6 kb and 5.8 kb are 5' coterminal and may both encode the novel glycoprotein gene EUS4. The 1.6 kb transcript terminates at a poly(A) signal site downstream of the EUS4 ORF, and the 5.8 kb transcript terminates within the inverted repeat (IR) segment. Overall, the transcriptional program of the EHV-1 KyA Us segment is complex and exhibits similarities to that of HSV-1 Us segment: a) transcripts arise from both DNA strands; b) some transcripts, including those mapping at the termini of the Us segment, extend into the IR segments and are 3' coterminal with the 1.2 kb IR6 transcript; c) at least one transcript reads through a functional polyadenylation signal; d) some transcripts encoding genes that lie in different reading frames exist as a family of overlapping mRNAs, some in an anti-sense manner. Lastly, of the six Us genes of the EHV-1 KyA strain, only those encoding the EHV-1 protein kinase and the HSV-2 gG/gX homolog are members of the early kinetic class.

Protection against lethal equine herpes virus type 1 (subtype 1) infection in hamsters by immune stimulating complexes (ISCOMs) containing the major viral glycoproteins

An experimental ISCOM vaccine has been prepared from gradient purified equine herpes virus type 1 (EHV-1). Radiolabelling studies demonstrated that this vaccine contained all the major viral glycoproteins in relative amounts similar to those found in non-detergent disrupted viral preparations. This EHV-1 ISCOM vaccine generated fully protective responses in hamsters challenged with an otherwise lethal dose of the hamster-adapted EHV-1 strain RACH.

Scanning Electron Microscope Reveals Striking Differences in Cytopathic Effects of Equine Herpesvirus (EHV) Strains

The phenomena of cytopathic effects (CPE) i.e., those virus-induced morphological changes in cultured cells observable at the level of light microscopy are commonly used in presumptive diagnosis of viral infections. The cytomorphological changes are similar for many viruses in the same Family or Genus. For example, in LLC-RK1 cell monolayers both EHV type 1-strain KyD (EHV-1) and EHV type 2-strain LK (EHV-2) cause focal cell rounding and polykaryocytosis which can be observed by phase-contrast microscopy (LM). To determine whether external and internal ultrastructural differences exist in LLC-RK1 cells (American Type Culture Collection # CCL 106) expressing CPE, cells were infected with each virus and processed for scanning electron microscopy (SEM).Monolayer cultures of LLC-RK1 cells on 12 mm glass coverslips were inoculated with EHV-1 or EHV-2 at low input multiplicity of infection. After an hour’s adsorption at 37 C, residual inoculum was removed, and cultures were washed twice with serum-free medium and incubated in growth medium at 37 C.