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.     

A deletion in the gI and gE genes of equine herpesvirus type 4 reduces viral virulence in the natural host and affects virus transmission during cell-to-cell spread

In order to identify the role of the equine herpesvirus type 4 (EHV-4) glycoprotein I (gI) and E (gE) genes in determining viral virulence and their affect on the infection cycle, we constructed an EHV-4 recombinant strain containing a deletion in both gI and gE genes and its revertant. The recombinant was assayed in vitro in order to compare its growth kinetics with the parent and revertant viruses. Our results indicated that a deletion in the genes encoding gI and gE affected cell-to-cell spread of the virus in vitro. In order to assess the pathogenicity and vaccine efficacy of the recombinant in a natural host, colostrum-deprived foals were inoculated intranasally with the recombinant. Clinical signs obtained in foals upon the inoculation with the recombinant were milder than that for the revertant. This suggests that intact gI and/or gE genes are important factors in the expression of virulence in EHV-4 as in seen in the case of other herpesviruses. In addition, full protection against challenge infection was observed in foals, which had undergone a previous inoculation of the recombinant.     

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.     

Biosynthesis and interaction of glycoproteins E and I of canine herpesvirus.

In cells infected with canine herpesvirus (CHV), the mature form of glycoprotein E (gE) had a molecular weight of 94 kDa, and that of glycoprotein I (gI) had a broad range of molecular weights of 55-62 kDa. gE and gI formed a complex like gE and gI of other alphaherpesviruses. When cells were infected with the gI minus mutant of CHV (gI/Z), the mature form of the 94 kDa gE was not formed, but a 76 kDa gE polypeptide was found. Similarly, no mature gI was formed in cells infected with the gE minus mutant of CHV (gE/Z), but a 40 kDa gI polypeptide was formed. When cells were coinfected with gE/Z and gI/Z, the molecular masses of gE and gI were increased from 76 to 94 kDa and from 40 to 55-62 kDa, respectively. We constructed vaccinia virus recombinants which expressed CHV gE or CHV gI. Only when cells were coinfected with both the vaccinia recombinant which expressed gE and the vaccinia recombinant which expressed gI, gE and gI were processed into their mature forms. Our results suggest that the presence of both gE and gI is necessary for efficient processing of the precursors of gE and gI to their mature forms.     

Characterisation of IE and UL5 gene products of equine herpesvirus 1 using DNA inoculation of mice

Summary.  The equine herpesvirus 1 (EHV-1) strain HVS25A regulatory genes IE and UL5, encoding homologues of herpes simplex virus 1 (HSV-1) ICP4 and ICP27 respectively, were cloned into a eukaryotic expression vector and the DNA injected intramuscularly into mice. Antibodies produced in this way detected the IE or UL5 gene products as diffuse material in nuclei of RK13 cells transfected with the individual genes but as discrete punctate or large aggregates in RK13 cells infected with EHV-1. Western blotting on EHV-1 infected RK13 cells showed multiple IE products of 120–200 kDa and a UL5 product of 52 kDa. Inoculation with plasmids expressing EHV-1 IE or UL5 provided limited protection against EHV-1 challenge in mice as determined by increased virus clearance from lungs on day 2 post-challenge and a reduction in severity of lung histopathology. However, this protection was relatively weak compared with that provided by inoculation of DNA encoding EHV-1 glycoprotein D (gD), possibly reflecting the importance of neutralising antibody in this model. Accepted May 19, 2000 Received March 20, 2000     

The extracellular part of glycoprotein E of bovine herpesvirus 1 is sufficient for complex formation with glycoprotein I but not for cell-to-cell spread

Summary.  Glycoproteins gE and gI of bovine herpesvirus 1 (BHV-1) are type I transmembrane proteins that can form a complex that is involved in cell-to-cell spread mechanisms. The extracellular domains of both proteins have cysteine-rich regions that are also found in the homologous proteins of other alphaherpesviruses. The extracellular domain of gE has two conserved cysteine-rich regions: C1 and C2. The other conserved regions in gE are located between C2 and transmembrane region and in the cytoplasmic domain of gE. We studied the complex formation between gE and gI using a series of truncated gE proteins and a full length form and a secreted form of gI. All proteins were expressed in recombinant baculoviruses. To analyse the complex formation between these polypeptides we used monoclonal antibodies (MAbs 67 and 75) that specifically react with the gE/gI complex and not with separately expressed glycoproteins gE and gI alone. This analysis showed that the BHV-1 gE/gI complex can be formed in insect cells after a co-infection with baculoviruses expressing gE and gI in their full length form. When secreted forms of gE and gI were expressed after co-infection, the gE/gI complex was still formed and could also be detected in the tissue culture medium. This gE/gI complex was also formed after mixing the tissue culture media of insect cells expressing the secreted form or gE or gI separately. The smallest part of gE that still formed a complex is encoded by the first 246 residues of gE. This extracellular domain contains only the C1 region, showing that the C2 region is not essential for gE/gI complex formation. Shorter forms of gE encoding the C1 region did not form a detectable complex. We also found that the formation of gE/gI complex is not sufficient for normal cell-to-cell spread of BHV-1. A recombinant BHV-1 gE TM-virus, expressing a truncated glycoprotein E from which the transmembrane and cytoplasmic domain were removed, forms plaques as small as a gE null mutant. Accepted July 26, 1999/Received March 5, 1999     

The C-terminal regions of the envelope glycoprotein gp2 of equine herpesviruses 1 and 4 are antigenically distinct

Summary.  The unusual mucin-like high molecular mass (Mr) glycoprotein 2 (gp2) has only been described in the equid alphaherpesviruses, among which there is considerable antigenic cross-reactivity. Equine herpesvirus 1 (EHV-1) gp2 is cleaved into a highly glycosylated N-terminal subunit and a 42 kDa C-terminal cleavage product. In order to investigate their antigenic recognition by horses naturally infected with EHV-1 and/or equine herpesvirus 4 (EHV-4), the C-terminal cleavage product and high Mr gp2 were affinity purified. Cross-reactivity between EHV-1 and EHV-4 was observed for the high Mr gp2 using Western blotting. In contrast only horses with antibodies to EHV-1 detected the 42 kDa EHV-1 gp2 C-terminal cleavage product. This phenomenon was evident in pooled sera from adult horses and also in foals that had demonstrated seroconversion due to EHV-1 infection. The results indicate that the C-terminal region of EHV-1 gp2 is antigenically distinct from that of EHV-4 gp2 and can be detected only after an EHV-1-specific immune response. Received June 23, 2001 Accepted November 7, 2001     

Epitopes on glycoprotein E and on the glycoprotein E/glycoprotein I complex of bovine herpesvirus 1 are expressed by all of 222 isolates and 11 vaccine strains

Summary.  Glycoprotein E (gE) of bovine herpesvirus 1 (BHV1) forms a complex with glycoprotein I (gI) and plays an important role in cell-to-cell spread mechanisms of the virus, but is not essential for propagation of the virus. To study the antigenic variability of BHV1 glycoprotein E, a set of six well characterised monoclonal antibodies (MAbs) was established using BHV1 gE and gI deletion mutants, eukaryotically expressed gE and gI and pepscan analysis. Two of these MAbs reacted with a linear gE epitope (MAbs 3 and 52), two reacted with a more conformation dependent gE epitope (MAbs 61 and 81) and two reacted with epitopes formed by a complex formed between gE and glycoprotein I (MAbs 67 and 75). With these six MAbs the gE expression of 222 BHV1 isolates and 11 BHV1 modified-live vaccine strains was studied in vitro, using an immunoperoxidase monolayer assay. All 222 BHV1 isolates and 11 vaccine strains were found to react with MAbs 61, 81 and 75. Three of the 222 isolates failed to react with MAb 67 and two of the vaccines reacted very weakly with MAbs 3 and 52. Analysis of the gE genes of these five aberrant isolates and the gE glycoproteins they expressed, did not show obvious size differences compared to wild-type BHV1. We conclude that the tested gE epitopes are highly conserved, including the epitopes formed by the gI/gE complex. Received September 15, 1999/Accepted December 16, 1999     

Spontaneous BHV1 recombinants in which the gI/gE/US9 region is replaced by a duplication/inversion of the US1.5/US2 region

Summary.  In a bovine herpesvirus 1 (BHV1) vaccine strain, a spontaneous BHV1 mutant (Za) was found that arose from a recombination between two isomeric forms of the BHV1 genome. In this Za mutant one end of the U_S region, containing part of the US1.5 gene, was found duplicated in an inverted orientation at the other end of the U_S region. Concurrently, a 2.7 kb deletion was found in Za that encompasses both the US8 (gE) and US9 gene. Analysis of the in vitro growth properties of a genetically modified BHV1gE⁻ mutant showed that at 11 hours post infection BHV1gE⁻ viruses were secreted ten times more efficiently than wild type virus. Using this observation we developed a protocol to enrich for spontaneous gE deletion mutants in a BHV1 field isolate and found another mutant (Rof3) with similar properties as the Za mutant. Rof3 has a duplication/inversion of the US1.5 gene and part of the US2 gene and a simultaneous 3.5 kb deletion that encompasses the US7 (gI), US8 (gE) and US9 genes. The nucleotide sequences of the recombination points of both recombinants were determined and compared. No obvious sequence similarities were found, suggesting that non-homologous recombination events led to the observed recombinations. The implications for the use of BHV1 gE deletion mutants as marker or diva vaccines are discussed. Received January 10, 1999 Accepted March 29, 1999     

The reactivity of monoclonal antibodies against orf virus with other parapoxviruses and the identification of a 39 kDa immunodominant protein

Summary.  A panel of 27 mouse monoclonal antibodies (Mabs) was raised against orf virus. Sixteen of these Mabs reacted with a protein with a molecular mass of 65 kDa, 8 reacted with a protein with a molecular mass of 39 kDa and three remain uncharacterised. Reactivity of the Mabs with a library of recombinant vaccinia viruses expressing various regions of the NZ-2 orf virus genome identified the approximate positions of the genes encoding these 2 immunodominant orf virus proteins. The gene encoding the 39 kDa protein was identified and sequenced. The protein was detected in an envelope fraction of orf virus and was shown to be homologous to the envelope protein encoded by the H3L gene of vaccinia virus. The 65 kDa protein has not been fully characterised, but the gene encoding it has been localised to a 10 kbp region of the orf virus genome. The Mabs were used to discriminate 4 parapoxviruses derived from sheep, 2 from cattle and 1 each from a seal and squirrel. Eighteen Mabs reacted with all 4 sheep viruses, 19 Mabs reacted with both cattle viruses, 6 recognised seal parapoxvirus and 2 recognised the squirrel parapoxvirus. Only one of the 27 Mabs reacted with all 8 parapoxviruses suggesting it recognises a conserved epitope within the genus. Received March 11, 1998 Accepted July 24, 1998     

Molecular phylogeny of equine herpesvirus 1 isolates from onager, zebra and Thomson’s gazelle

Viruses related to equine herpesvirus type 1 (EHV-1) were isolated from an aborted fetus of an onager (Equus hemionus) in 1984, an aborted fetus of Grevy’s zebra (Equus grevyi) in 1984 and a Thomson’s gazelle (Gazella thomsoni) with nonsuppurative encephalitis in 1996, all in the USA. The mother of the onager fetus and the gazelle were kept near plains zebras (Equus burchelli). In phylogenetic trees based on the nucleotide sequences of the genes for glycoproteins B (gB), I (gI), and E (gE), and teguments including ORF8 (UL51), ORF15 (UL45), and ORF68 (US2), the onager, Grevy’s zebra and gazelle isolates formed a genetic group that was different from several horse EHV-1 isolates. Within this group, the onager and gazelle isolates were closely related, while the Grevy’s zebra isolate was distantly related to these two isolates. The epizootiological origin of the viruses is discussed.     

Varicella-Zoster Virus Glycoproteins E and I Expressed in Insect Cells Form a Heterodimer That Requires the N-Terminal Domain of Glycoprotein I

Varicella-zoster virus (VZV) glycoproteins E and I (gE and gI), which are major components of the virion envelope, form a noncovalently linked complex. To understand their properties and functions, we expressed and purified soluble forms of gE and gI in the baculovirus system. Extracellular domains of gE and gI were cloned into baculoviruses, using either native or insect-derived signal peptides. Each recombinant virus yielded soluble protein in culture medium although a higher level of secretion was achieved with insect-derived signal peptides in recombinant gE baculoviruses. A soluble gE–gI complex was formed by co-infecting insect cells with recombinant gE and gI baculoviruses and detected by immunoprecipitation followed by Western blotting analyses. By gel filtration and cross-linking studies, we showed that the VZV gE–gI complex expressed in insect cells is a heterodimer. Interestingly, two recombinant gI proteins in which signal peptides were replaced with insect-derived signal peptides did not associate with gE. Amino-terminal sequencing and site-specific mutational studies showed that the replacement of only the signal peptides did not prevent complex formation but alterations in the processed amino-terminus of gI abrogated its ability to complex with gE. These findings indicate that the mature amino-terminus of gI is required for gE–gI complex formation by the external domains of VZV gE and gI.     

Host cell tropism of equine herpesviruses: glycoprotein D of EHV-1 enables EHV-4 to infect a non-permissive cell line

Summary 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.     

Herpes simplex virus IgG Fc receptors induced using recombinant adenovirus vectors expressing glycoproteins E and I

Evidence has been presented that herpes simplex virus (HSV) immunoglobulin (IgG) Fc receptors are composed of a complex of two glycoproteins, gE and gI. In previous studies, cells infected with HSV-1 mutants lacking either gE or gI bound lower levels of soluble IgG than cells infected with wild-type viruses suggesting that both gE and gI were required for IgG binding. We have reevaluated the Fc receptor activity of these mutants using a more sensitive assay involving IgG-coated erythrocytes and have found that cells infected with a gE- mutant HSV-1 did not bind IgG-coated erythrocytes whereas cells infected with a gI- mutant retained some Fc binding activity. To further study HSV-induced Fc receptors recombinant adenovirus vectors expressing gE or gI were constructed. Cells expressing gE alone bound both soluble IgG and IgG-coated red cells, although the binding was consistently lower than that observed with HSV-infected cells or cells expressing both gE and gI. Cells expressing only gI were unable to bind either soluble IgG or IgG-coated erythrocytes. These results support the conclusion that both gE and gI are required for full Fc receptor activity, although gE alone can bind IgG to a lesser extent.     

Characterization of changes in the short unique segment of pseudorabies virus BUK-TK900 (Suivac A) vaccine strain

Summary. Mutant strains of pseudorabies virus (PRV) of reduced virulence, such as Bartha or BUK-TK900, have been used for vaccination purposes for many years. In contrast to the Bartha strain, BUK-TK900 has not been well characterised at the molecular level. The detailed analysis of this vaccine strain was urged by the fact of the isolation in Poland of field strains which were suspected to originate from BUK-TK900.We characterised changes in the U_S region of this strain, focusing our attention on gE and gI genes. The only deletion, about 300bp, found in BamHI 7 fragment (covering most of the U_S region) was located in the 28K (US2) gene. BUK-TK 900 produced small plaques on all cell lines tested in our laboratory (SK6, Vero, MDBK, 3T3). The plaque size was restored to about 70% of wild type virus plaque size when growing BUK-TK900 virus on 3T3 complementing cell line expressing PRV gE and up to 100% when cell line producing gE and gI was used. Both gE and gI genes from BUK-TK900 and from some derivative field isolates have been amplified by PCR reaction but no deletions in these genes have been found. Molecular weight of gene products differed from wild type proteins: gE was bigger than wild type gE while gI was smaller. Both proteins were correctly recognised by all tested polyclonal and monoclonal antibodies. Radioimmunoprecipitation study showed that BUK-TK900 gE and gI interact forming a complex. The whole ORF of BUK-TK900 gE was sequenced and only few point mutations were found; only two of them led to changes of amino acids in the polypeptide chain. These were: methionine at position 124 replaced by threonine and glutamine at position 162 replaced by arginine. The introduction of first of these mutations (Met to Thr) to PRV wild type strain NIA-3 resulted in 22% reduction of plaque size. This result confirms the importance of this domain of gE for its function; it was found previously by others that deletion of amino acids 125 and 126 reduced virulence and neurotropism of PRV. More changes were found in BUK-TK900 gI sequence. Over 80% of these changes were located in the terminal 1/3rd of the sequence. Some of these mutations may have significant effect on the secondary structure of gI glycoprotein. The change of the secondary structure may be responsible for the decrease of gI stability and the observed reduction of gI molecular mass.Present address: Cedi-Diagnostics B.V., Lelystad, The Netherlands.Received May 4, 2001; accepted March 18, 2003 Published online June 11, 2003     

Cloning of the genome of equine herpesvirus 4 strain TH20p as an infectious bacterial artificial chromosome

Equine herpesvirus 4 (EHV-4) is a major cause of respiratory tract disease in horses worldwide. The generation of recombinant viruses, which would lead to understanding of viral gene functions, has been hindered by the absence of suitable cell lines and small-animal models of the infection. In the present study, the genome of EHV-4 strain TH20p was cloned as a stable and infectious BAC without any deletions of the viral genes. Mini F plasmid sequences flanked by loxP sites were inserted into the intergenic region between genes 58 and 59. Coinfection of the recombinant virus with a recombinant adenovirus expressing Cre recombinase resulted in the excision of the BAC sequences. Importantly, the resulting recombinant EHV-4 replicated comparably to the wild-type virus in fetal horse kidney cells. The recombinant EHV-4 will facilitate EHV-4 research and provide the opportunity to exploit the power of BAC technology for production of recombinant viral vaccines.     

Equine herpesvirus 1 (EHV-1) glycoprotein M: effect of deletions of transmembrane domains

Equine herpesvirus 1 (EHV-1) recombinants that carry either a deletion of glycoprotein M (gM) or express mutant forms of gM were constructed. The recombinants were derived from strain Kentucky A (KyA), which also lacks genes encoding gE and gI. Plaques on RK13 cells induced by the gM-negative KyA were reduced in size by 80%, but plaque sizes were restored to wild-type levels on gM-expressing cells. Electron microscopic studies revealed a massive defect in virus release after the deletion of gM in the gE- and gI-negative KyA, which was caused by a block in secondary envelopment of virions at Golgi vesicles. Recombinant KyA expressing mutant gM with deletions of predicted transmembrane domains was generated and characterized. It was shown that mutant gM was expressed and formed dimeric and oligomeric structures. However, subcellular localization of mutant gM proteins differed from that of wild-type gM. Mutant glycoproteins were not transported to the Golgi network and consequently were not incorporated into the envelope of extracellular virions. Also, a small plaque phenotype of mutant viruses that was indistinguishable from that of the gM-negative KyA was observed. Plaque sizes of mutant viruses were restored to wild-type levels by plating onto RK13 cells constitutively expressing full-length EHV-1 gM, indicating that mutant proteins did not exert a transdominant negative effect on wild-type gM.     

Formation of Pseudorabies virus glycoprotein E/I complex in baculovirus recombinant system

Glycoproteins E (gE) and I (gI) of Pseudorabies virus (PRV) form a non-covalently bound complex to which a number of functions have been attributed. The gE/gI complex formation was studied using a series of full-length and truncated forms of gE and gI expressed in baculovirus recombinant system. Both glycoproteins were truncated by stepwise removal of their C-terminal parts and their ability to form the complex was studied by radioimmunoprecipitation. It was found that N-terminal domains of gE and gI containing first 122 and 106 aa, respectively, were sufficient for the complex formation.