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.     

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.     

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.     

Cloning of the herpes simplex virus type 1 genome as a novel luciferase-tagged infectious bacterial artificial chromosome

Herpes simplex virus type 1 (HSV-1) is a ubiquitous human pathogen of skin and mucous membranes. In the present study, the genome of the HSV-1 F strain was cloned as an infectious bacterial artificial chromosome (BAC) clone without any deletions of the viral genes. Additionally, a firefly luciferase cassette was inserted to generate a novel luciferase-expressing HSV-1 BAC. Importantly, the resulting recombinant HSV-1 BAC Luc behaved indistinguishably from the wild-type virus in Vero cells, and the luciferase activity could be easily quantified in vitro. Thus, this novel HSV-1 BAC system would serve as a powerful tool for gene function profiling.     

Development of a bacterial artificial chromosome (BAC) recombineering procedure using galK-untranslated region (UTR) for the mutation of diploid genes

Bacterial artificial chromosome (BAC) recombineering using galK selection allows DNA cloned in Escherichia coli to be modified without introducing an unwanted selectable marker at the modification site. Genomes of some herpesviruses have a pair of inverted repeat sequences that makes it very difficult to introduce mutations into diploid (duplicate) genes using the galK selection method. To mutate diploid genes, we developed a galK-UTR BAC recombineering procedure that blocks one copy of the target diploid gene by insertion of a galK untranslated region (UTR), which enables the simple mutation of the other copy. The blocked copy can then be replaced with an UTR-specific primer pair. The IR2 gene of equine herpesvirus 1 (EHV-1) maps within both the internal (IR) and terminal repeat (TR) of the genomic short region and is expressed at low levels because its promoter is TATA-less. Both IR2 promoters in EHV-1 BAC were replaced with a mutant IR2 promoter containing three Sp1-binding motifs and a consensus TATA box by galK-UTR BAC recombineering. The expression level of the IR2 protein controlled by the modified promoter increased approximately 4-fold as compared to that of wild-type EHV-1. The galK-UTR method will provide a useful tool in studies of herpesviruses.     

Molecular cloning of the guinea pig cytomegalovirus (GPCMV) genome as an infectious bacterial artificial chromosome (BAC) in Escherichia coli

Since cytomegalovirus (CMV) infection is highly species-specific, it is necessary to study animal cytomegaloviruses to assess viral factors which contribute to pathogenesis. The generation of recombinant viruses carrying reporter genes would provide useful tools for studying the genetics of CMV pathogenicity in vivo. We evaluated whether the guinea pig cytomegalovirus (GPCMV) was amenable to such manipulation. Metabolic selection using the guanosylphosphoribosityl transferase (gpt) gene facilitated recovery of a recombinant virus, vAM403, containing a gpt/green fluorescent protein (eGFP) cassette introduced into the HindIII "N" region of the viral genome. This virus had replication kinetics identical to wild-type virus. We next attempted to clone the GPCMV genome as a bacterial artificial chromosome (BAC). A BAC plasmid containing a gpt/eGFP cassette and the chloramphenicol resistance marker was introduced into HindIII "N" to generate another GPCMV recombinant, vAMBGPCMV. Circular viral DNA isolated from vAMBGPCMV-infected cells was used to transform Escherichia coli. Restriction profiles revealed that the GPCMV genome had been cloned as a BAC plasmid, and transfection of BAC plasmid DNA confirmed that the BAC clone was infectious. A novel strategy based on a unique PmeI site was devised to quickly modify the BAC GPCMV plasmid. Recombinants retained the capability to replicate and express reporter genes in guinea pigs, suggesting that these viruses will be useful for in vivo pathogenesis studies.     

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.     

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.     

Identification of essential and non-essential genes of the guinea pig cytomegalovirus (GPCMV) genome via transposome mutagenesis of an infectious BAC clone

We report application of a transposition methodology that allows the easy characterization and mutation of genes encoded on an infectious bacterial artificial chromosome (BAC) clone. We characterized mutants generated by transposome (Tn) mutagenesis of a BAC clone of guinea pig cytomegalovirus (GPCMV). A pool of Tn mutant GPCMV BACs were screened initially by restriction profile analysis to verify they were full-length, and subsequently GPCMV BAC DNA from individual mutants was transfected onto guinea pig lung fibroblast cells in order to generate virus. Tn GPCMV BAC mutants were classed as either essential or non-essential gene insertions, depending upon their ability to regenerate viable, replication-competent virus. Representative mutants were more fully characterized. Analysis by sequencing the Tn insertion site on the mutated BACs, and by regeneration of virus using transfection of guinea pig fibroblasts (GPL), demonstrated that a recombinant with a Tn insertion in the UL35 homolog gene (GP35) was a non-essential gene for viral replication in tissue culture. A mutant with an insertion in the UL46 homolog (GP46) was nonviable, a phenotype which could be rescued by homologous recombination of BAC DNA with wild-type UL46 sequences, suggesting an essential role of this putative capsid gene in virus replication.     

Equine herpesvirus in vivo: cyclic production of a DNA density variant with repetitive sequences.

The DNA of a strain of equine herpesvirus type 1 passed more than 500 times in Syrian hamsters (EHV-1ha) has been analyzed by CsCl equilibrium density gradient ultracentrifugation, analytical sedimentation, and DNA-DNA reassociation kinetics. The viral DNA consisted of light and heavy species having densities in CsCl of 1.716 and 1.724 g/cm³, which correspond to guanine plus cytosine contents of 56 and 64%, respectively. These values were confirmed by T_m measurements. Similar molecular weight values were obtained by analytical sedimentation for the light (87.9 × 10⁶) and heavy (81.8 × 10⁶) DNA species. The heavier species was produced in a cyclic manner. Hamsters infected with virus containing a high proportion of the heavy species gave reduced virus yields and survived longer. The genetic relatedness of the two viral DNA species of EHV-1ha was compared by examining the ability of each to reanneal with ³²P-labeled viral DNA of the tissue culture strain (L-M cell) of EHV-1 (EHV-1tc). The lighter (1.716 g/cm³) species of EHV-1ha was composed of unique sequences completely homologous to the entire EHV-1tc genome, while the heavier species (1.724 g/cm³) consisted of sequences homologous to approximately 50% of the EHV-1tc genome. Of these homologous sequences, 40–60% (20–30% of the entire EHV-1tc genome) were reiterated. Further, analyses of the EHV-1tc genome (fragmented and unfragmented) by thermal chromatography on hydroxylapatite and in neutral preparative CsCl equilibrium density gradients revealed considerable intramolecular heterogeneity in nucleotide distribution. Finally, analysis of the structural polypeptides of virions of EHV-1ha which contained the heavy and light DNA species revealed that the following two major viral proteins were missing from virions containing the heavier DNA species: VP8, an envelope protein with a molecular weight of 173,000, and VP23, a nucleocapsid protein with a molecular weight of 38,000.     

Properties of an equine herpesvirus 1 mutant devoid of the internal inverted repeat sequence of the genomic short region

The 150 kbp genome of equine herpesvirus-1 (EHV-1) is composed of a unique long (UL) region and a unique short (Us) segment, which is flanked by identical internal and terminal repeat (IR and TR) sequences of 12.7 kbp. We constructed an EHV-1 lacking the entire IR (vL11ΔIR) and showed that the IR is dispensable for EHV-1 replication but that the vL11ΔIR exhibits a smaller plaque size and delayed growth kinetics. Western blot analyses of cells infected with vL11ΔIR showed that the synthesis of viral proteins encoded by the immediate-early, early, and late genes was reduced at immediate-early and early times, but by late stages of replication reached wild type levels. Intranasal infection of CBA mice revealed that the vL11ΔIR was significantly attenuated as mice infected with the vL11ΔIR showed a reduced lung viral titer and greater ability to survive infection compared to mice infected with parental or revertant virus.     

Deletion of the UL4 gene sequence of equine herpesvirus 1 precludes the generation of defective interfering particles

Serial, high multiplicity passage of equine herpesvirus 1 (EHV-1) leads to the generation of defective interfering particles (DIP). EHV-1 DIP inhibit and interfere with the replication of standard EHV-1, establishing a state of persistent infection. These DIP package severely truncated and rearranged forms of the standard viral genome. Contained within the DIP genome are only three genes: UL3, UL4, and a unique hybrid gene (Hyb). The hybrid gene forms through a recombination event that fuses portions of the early regulatory IR4 and UL5 genes and is essential for DIP-mediated interference. The UL4 gene is an early gene dispensable for lytic replication and inhibits viral and cellular gene expression. However, the contribution of the UL4 gene during DIP-mediated persistent infection is unknown. Here, we describe the generation of a completely deleted UL4 virus and its use to investigate the role of the UL4 gene in the generation of the defective genome. Deletion of the UL4 gene resulted in delayed virus growth at late times post-infection. Cells infected with a mutant EHV-1 that lacked expression of the UL4 protein due to an inserted stop codon in the UL4 gene produced defective particles, while cells infected with a mutant EHV-1 that had the complete UL4 gene sequence deleted were unable to produce DIP. These data suggest that the UL4 gene sequence, but not the UL4 protein, is critical for the generation of defective interfering particles.     

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.     

Identification of the products of the equine herpesvirus type 4 gI and gE genes

Summary.  In order to identify the products of the equine herpesvirus type 4 (EHV-4) gI and gE genes, we have constructed recombinant vaccinia viruses containing the putative gI or gE genes. These recombinant viruses synthesized EHV-4 gI and gE with apparent molecular masses of 75 and 80 kDa, respectively. Antibodies raised against both recombinant viruses detected a 75 kDa gI and a 95 kDa gE in EHV-4-infected cells. The results also suggest that the EHV-4 gI and gE would form a complex like in other herpesviruses. Received October 29, 1999 Accepted January 21, 2000     

Live-attenuated recombinant equine herpesvirus type 1 (EHV-1) induces a neutralizing antibody response against West Nile virus (WNV)

The immunogenicity in horses of a recombinant equine herpesvirus type 1 (EHV-1) vaccine expressing West Nile virus (WNV) prM and E proteins was studied. To construct the recombinant EHV-1, two-step en passant mutagenesis was employed for manipulation of a bacterial artificial chromosome (BAC) of vaccine strain RacH. Recombinant EHV-1 stably expressed the WNV prM and E proteins as demonstrated by indirect immunofluorescence and Western blotting. In addition, growth properties in vitro of the EHV-1/WNV recombinant were found to not be significantly different from those of the parental virus. To determine if vaccination of horses induces an antibody response, 10 horses were allocated in two groups. Group A consisted of six horses that were vaccinated three times with the recombinant EHV-1/WNV virus in 28- to 31-day intervals. Group B consisted of four horses that were sham-vaccinated using the same regimen. Serum was collected on days 0, 31, 45 and 66. Plaque reduction neutralization test and IgG(T)- and IgGb-specific WNV E antibody-capture ELISAs were used. After a single vaccination (day 31), at least four of the six horses from group A had detectable levels of serum neutralizing antibodies against WNV, and three horses retained SN titers until the end of the study. None of the horses in the control group B sero-converted. On days 31 and 45, five of the six horses in group A had a marked increase of WNV-specific IgG(T), and at least four exhibited modestly elevated WNV-specific IgGb titers. From the results, we concluded that the EHV-1 vectored virus is able to express the WNV structural proteins and that vaccination of horses results in the induction of WNV E-protein-specific IgG(T), IgGb, and neutralizing antibodies.     

Construction and characterization of bacterial artificial chromosomes containing HSV-1 strains 17 and KOS

Bacterial artificial chromosomes (BACs) were constructed containing full-length, infectious DNA of HSV-1 strains 17 and KOS. To generate BACs without altering viral genes, sequences required for selection and propagation of the BAC were placed between the U(L)37 and U(L)38 genes, and flanked by LoxP sites. The system was tested by studying multiple properties of these HSV-1 BAC constructs in vitro and in vivo following propagation in bacteria, virus reconstitution from HSV-BAC DNA in eukaryotic cells, and Cre-recombinase-mediated excision of the BAC backbone. Based on in vitro growth in mouse embryo fibroblasts and in vivo growth in mouse corneas and trigeminal ganglia, the strain KOS BAC-derived virus behaved similarly to wild-type. Small changes in neurovirulence were, however, observed. The strain 17 BAC-derived virus exhibited modest decreases in growth and virulence compared to wild-type. Modest differences were observed in reactivation from latency with both strain KOS and 17 BAC-derived viruses. In addition, the system was further validated by performing mutagenesis of the BACs by allelic exchange in E. coli. These BACs are suitable for the rapid generation of recombinant viruses for pathogenesis and other studies, but as with all mutagenesis systems, care must be taken in their construction and repair.     

Promoter activity in the 5′ flanking regions of the Bohle iridovirus ICP 18, ICP 46 and major capsid protein genes

Summary. Bohle iridovirus (BIV) belongs to the genus Ranavirus, of which Frog virus 3 (FV-3) is the type species. We are developing BIV as a recombinant viral delivery vector, and as a first step we located specific BIV promoter sequences to drive foreign gene expression in the recombinant virus. By comparison with FV-3 sequences, the genes encoding ICP 18 and ICP 46 in BIV were identified and sequenced. Putative promoter regions of these two early genes and of the major capsid protein (MCP) gene were identified, cloned into pSFM21, and luciferase production was then used to assess the promoter activity of these regions.