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.     

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.     

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 (H Delta gp2) reconstituted from pRacH lacked gene 71 and did not express gp2 as assayed by indirect immunofluorescence analysis using gp2-specific monoclonal antibodies. The H Delta gp2 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 (H Delta gp2gM) did not express either of the deleted glycoproteins as demonstrated by indirect immunofluorescence analysis. The H Delta gp2gM 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 H Delta gp2 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.     

Identification and characterization of the pseudorabies virus UL43 protein

Among the least characterized herpesvirus membrane proteins are the homologs of UL43 of herpes simplex virus 1 (HSV-1). To identify and characterize the UL43 protein of pseudorabies virus (PrV), part of the open reading frame was expressed in Escherichia coli and used for immunization of a rabbit. The antiserum recognized in Western blots a 34-kDa protein in lysates of PrV infected cells and purified virions, demonstrating that the UL43 protein is a virion component. In indirect immunofluorescence analysis, the antiserum labeled vesicular structures in PrV infected cells which also contained glycoprotein B. To functionally analyze UL43, a deletion mutant was constructed lacking amino acids 23-332 of the 373aa protein. This mutant was only slightly impaired in replication as assayed by one-step growth kinetics, measurement of plaque sizes, and electron microscopy. Interestingly, the PrV UL43 protein was able to inhibit fusion induced by PrV glycoproteins in a transient expression-fusion assay to a similar extent as gM. Double mutant viruses lacking, in addition to UL43, the multiply membrane spanning glycoproteins K or M did not show a phenotype beyond that observed in the gK and gM single deletion mutants.     

Functional characterization of EUL47 in productive replication, morphogenesis and infectivity of equine herpesvirus 1

EUL47 is a major component of the tegument of equine herpesvirus 1 (EHV-1). To determine its function, we used Red/ET cloning to delete its gene (gene 13) from EHV-1 strain Ab4p inserted into a bacterial artificial chromosome (BAC), yielding Ab4pattBΔ13. We also examined the reverted virus (Ab4pattB13R). Ab4pattBΔ13 replicated in rabbit kidney (RK)-13 cells, indicating that ORF13 is dispensable for virus replication in cell culture. Its intracellular and extracellular titers were about 10- and 100-fold lower than those of the revertant and parent strains, respectively. In addition, the plaque size was half the plaque sizes of the other two strains. The particle-to-plaque forming unit ratio of Ab4pattBΔ13 was 21-fold greater than the ratios of the revertant and parent strains. No enveloped virions were detected in the cytoplasm of Ab4pattBΔ13-infected cells by transmission electron microscopy. In hamster, Ab4pattBΔ13 caused clinical signs and weight loss after only 1 day, but induced less severe neurological signs than did the revertant and parent strains. These results indicate that EUL47 is structurally required for normal virus replication, viral morphogenesis and viral infectivity, and that loss of EUL47 moderately attenuates the neuropathogenicity of EHV-1 in the hamster model.     

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.     

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 M(r) 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 (gamma-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 (LDelta45 and HDelta45) 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 type 1 (EHV-1) glycoprotein K is required for efficient cell-to-cell spread and virus egress

The function of the equine herpesvirus type 1 (EHV-1) glycoprotein K (gK) homologue was investigated. Deletion of 88% of the UL53-homologous open reading frame in EHV-1 strain RacH resulted in a severe growth defect of the gK-negative virus (HDeltagK) as reflected by a significant decrease in the production of infectious virus progeny on RK13 cells. The HDeltagK virus induced only minute plaques, was unable to form syncytia, and its penetration efficiency into RK13 cells was reduced by approximately 40%. To further analyze gK function and intracellular trafficking, gK of strain RacH was replaced by a C-terminally truncated gK-green fluorescent protein fusion protein (gK-GFP). The generated recombinant virus was shown to replicate well on non-complementing cells, and virus penetration and syncytium formation were comparable to parental RacH. A reduction in plaque size and slightly decreased intra- and extracellular virus titers, however, were observed. The gK-GFP fusion protein was expressed with early-late kinetics, and multiple forms of the protein exhibiting M(r)s between 50,000 and 85,000 were detected by Western blot analysis. The various gK-GFP forms were shown to be N-glycosylated, associated with membranes of the Golgi apparatus, and were incorporated into extracellular virions. Complete processing of gK-GFP was only observed within the context of viral infection. From the results, we concluded that EHV-1 gK is required for efficient virus growth in vitro and that the carboxy-terminal amino acids are not required for its function, because the gK-GFP fusion protein was able to complement for EHV-1 growth in the absence of authentic gK.     

EHV-1 glycoprotein D (EHV-1 gD) is required for virus entry and cell-cell fusion, and an EHV-1 gD deletion mutant induces a protective immune response in mice

Summary.  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 (ΔgD 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 ΔgD 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 ΔgD 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 ΔgD 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 ΔgD 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. Received January 14, 2000/Accepted April 27, 2000     

In vitro and in vivo characterization of equine herpesvirus type 1 (EHV-1) mutants devoid of the viral chemokine-binding glycoprotein G (gG)

Glycoprotein G (gG) of equine herpesvirus type 1 (EHV-1), a structural component of virions and secreted from virus-infected cells, was shown to bind to a variety of different chemokines and as such might be involved in immune modulation. Little is known, however, about its role in the replication cycle and infection of EHV-1 in vivo. Here we report on the function of gG in context of virus infection in vitro and in vivo. A gG deletion mutant of pathogenic EHV-1 strain RacL11 (vL11DeltagG) was constructed and analyzed. Deletion of gG had virtually no effect on the growth properties of vL11DeltagG in cell culture when compared to parental virus or a rescuant virus vL11DeltagGR, respectively, and virus titers and plaque formation were unaffected in the absence of the glycoprotein. Similarly, in the murine model of EHV-1 infection, no significant differences in virulence between the gG deletion mutant and RacL11 or vL11DeltagGR were found at high doses of infection. However, infection of mice at lower doses revealed that the gG deletion mutant was able to replicate to higher titers in lungs of infected mice. Additionally, these mice lost significantly more weight than those infected with RacL11 and a more pronounced inflammatory response in lungs was observed. Therefore we concluded that deletion of gG in EHV-1 seems to lead to an exacerbation of respiratory disease in the mouse.     

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.     

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.     

In vitro transposon mutagenesis of an equine herpesvirus 1 genome cloned as a bacterial artificial chromosome

Summary. The 150-kbp genome of the alphaherpesvirus equine herpesvirus 1 (EHV-1) strain HVS25A was cloned as a bacterial artificial chromosome (EHV-1 BAC), with mini F plasmid sequences inserted between genes 62 and 63. Transfection of EHV-1 BAC DNA purified from E. coli gave rise to progeny virus that had a similar growth rate and yield in mammalian cell culture to those of parental wild-type EHV-1. Using in vitro mutagenesis with a Mu transposon, a large library of EHV-1 BAC mutants was generated, and sequence analysis indicated that insertions were dispersed randomly across the EHV-1 genome. Following transfections of a pilot sample of mutant EHV-1 BAC DNAs into mammalian cells, no CPE was observable by light microscopy for mutants carrying insertions in genes for the major capsid protein, large tegument protein, glycoprotein K, catalytic subunit of DNA polymerase, or single-stranded DNA-binding protein. Mutants that were able to produce CPE similar to wild-type EHV-1 included those with interruptions in ORFs of several tegument proteins. Analysis of several glycoprotein gene mutants indicated that those carrying insertions near the start of genes encoding glycoproteins E and I were viable, but showed markedly diminished plaque areas. These results were supported by confocal microscopy of transfected or infected cultures. Electron microscopy of cells infected with a gE mutant revealed accumulations of particles within cytoplasmic vesicles, consistent with a partial obstruction of maturation. The transposon library is a resource for comprehensive functional analysis of the HVS25A genome, with multiple mutants available in any of the predicted genes of EHV-1.     

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.     

In vitro characterization of EHV-4 gG-deleted mutant

Equine herpesvirus 4 (EHV-4) is an important pathogen that causes respiratory tract disease in horse populations worldwide. Glycoprotein G (gG) homologs have been identified in several alphaherpesviruses as minor non-essential membrane-anchored glycoproteins. In this study, EHV-4 gG deletion mutant has been generated by using bacterial artificial chromosome technology to investigate the role of gG in viral pathogenesis. Our findings reported here revealed no significant difference between parental EHV-4 and gG-negative strain in their replication cycle in cell culture. Furthermore, virus titers and plaque formation were comparable in both viruses. It is noteworthy that these findings disagree with the previously published study describing gG deletion in another EHV-4 strain.     

Pathogenicity of glycoprotein C negative mutants of herpes simplex virus type 1 for the mouse central nervous system

A previous study from our laboratory showed that a mutant of herpes simplex virus type 1 (HSV-1), strain KOS-321, carrying a deletion in the structural gene for glycoprotein C (gC) had reduced pathogenicity for the mouse central nervous system when compared to the wild-type virus (Kümel et al., 1985). In this study, eight additional gC negative (gC-) mutants derived from KOS-321 were shown to vary widely in their ability to induce lethal encephalitis in female DBA/2 mice following intracerebral inoculation. This variation in virulence showed no correlation with thymidine kinase activity. One less virulent gC- strain, gC-39, was further studied to determine whether the neurovirulent phenotype could be restored by rescue of the gC gene using standard marker rescue cotransfection procedures. The resulting progeny contained 2% gC+ recombinant virions and was tested for its ability to cause encephalitis. Although this progeny had increased virulence, it was not attributable to the acquisition of the gC gene since passive immunization of mice with a pool of anti-gC monoclonal antibodies had no effect on the development of encephalitis and only gC- viruses were isolated from diseased brain tissues. In agreement with these findings, individual plaque-purified gC positive (gC+) virus recombinants were shown not to have been restored to the wild-type virus level of neurovirulence. It is concluded that gC is not a virulence determinant in this mouse model of HSV-induced encephalitis and that cotransfection procedures can induce additional mutations that affect viral pathogenesis.     

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