Construction and characterization of a herpes simplex virus type I recombinant expressing green fluorescent protein: acute phase replication and reactivation in mice

A recombinant HSV-1 virus expressing EGFP from the HCMV major immediate early promoter (KOS-CMVGFP) was constructed to monitor viral replication and spread in vitro and in mice. KOS-CMVGFP replicated as efficiently as wild-type virus, strain KOS, in single cycle growth experiments in Vero cells indicating that the recombinant virus has no significant growth defects in vitro. Following ocular inoculation of mice, KOS-CMVGFP exhibited slight but statistically significant reductions in mouse tear film titers relative to wild-type virus. Progression of virus infection of the eyes, periocular tissue, and snout was readily followed by fluorescence microscopy. Insertion of the EGFP expression cassette into the KOS genome had no effect on the efficiency of establishment of latency as determined by quantitative competitive PCR of viral genomes in latently infected TG. KOS-CMVGFP reactivated with wild-type kinetics and efficiency by explant cocultivation, but exhibited a significant delay in the kinetics and a modest reduction in the efficiency of reactivation compared to KOS in the more sensitive TG cell culture model. Notably, EGFP expression preceded the detection of infectious virus by greater than 24 h in both ex vivo models and thus is a useful marker of the early stages in the induction of reactivation.     

Characterization of encephalitis in adult mice induced by intracerebral inoculation of herpes simplex virus type 1 (KOS) and comparison with mutants showing decreased virulence

The spread of herpes simplex virus type 1 (HSV-1) strain KOS, and two less neurovirulent mutants of the strain was studied in female DBA/2 mice during the 1- to 5-day postinoculation period after intracerebral inoculation. Immunohistopathology showed that wild-type KOS virus first infected the meninges and ependymal cells but did not infect cells at the inoculation sites. The virus continued to spread to some cells directly adjacent to ventricles; however, the most extensive and severe lesions were found in the pyriform lobes and other structures associated with the limbic system. The pattern of spread suggested that direct cell to cell viral spread is important but that retrograde axonal transport to distant sites probably accounts for the more severe lesions associated with the limbic system. Both less neurovirulent mutant viruses multiplied to a much lesser degree in the brain and spread less extensively than the wild type virus when equivalent doses were given; however, when a large dose of the least neurovirulent mar C10.1 mutant virus was inoculated, infection spread rapidly to the same regions of the brain affected by KOS. Studies of mar C10.1 showed that thymidine kinase deficiency, rather than a mutation in the gene coding for glycoprotein C, probably accounted for the decreased neurovirulence of this mutant. This mouse model of HSV-1 virus-induced encephalitis, in combination with appropriate studies of the molecular biology of the HSV-1 KOS strain, should be useful for the study of neurovirulence factors contributing to the pathogenesis of HSV-1.     

Thymidine kinase of herpes simplex virus type 1 strain KOS lacks mutator activity

The effect of thymidine kinase (TK) encoded by herpes simplex virus type 1(HSV-1) strain KOS in DNA replication fidelity was examined by two different mutagenesis assays. Mutagenesis assay of the LacZ reporter gene present in recombinant tkLTRZ1, which contained the integrated LacZ gene in the tk locus, revealed a less than 0.05% mutation frequency of the LacZ gene regardless of whether the viruses were propagated in TK-expressing cells or control cells, conflicting an earlier report that a HSV-1 TK(+) strain replicated a 0.5% mutation frequency of the LacZ gene (R. B. Pyles and R. L. Thompson, 1994, J. Virol. 68, 4514-4524). Furthermore, TK-proficient and -deficient recombinant viruses replicated with similar mutation frequencies (0.027 and 0.026%, respectively) of the LacZ gene, which was integrated in the polymerase locus. Results of SupF mutagenesis assay demonstrated that neither the spectra of mutation nor the mutation frequencies of SupF gene, which was integrated in the tk locus of recombinant, were significantly different (P > 0.05) in progeny viruses grown in TK-expressing cells and control cells. Therefore, both LacZ and SupF mutagenesis assays demonstrated that TK of the HSV-1 strain KOS did not have detectable mutator activity.     

Transgenic reporter mouse for bioluminescence imaging of herpes simplex virus 1 infection in living mice

Bioluminescence imaging allows spatial and temporal progression of viral infection to be detected and quantified in living mice, thereby providing a new approach for studies of viral-host pathogenesis. It has been necessary to construct and validate recombinant reporter viruses that express firefly luciferase to investigate viral replication and spread with this imaging technology. This strategy greatly limits the ability to analyze multiple strains of virus and/or existing viral mutants, and reporter viruses also may be attenuated relative to the respective parental viruses. To facilitate bioluminescence imaging of herpes simplex virus type 1 (HSV-1), we developed a transgenic reporter mouse that uses the promoter from HSV-1 thymidine kinase to control expression of firefly luciferase. Infection with HSV-1 activated expression of firefly luciferase in corneal and flank models of infection, and amounts of bioluminescence increased in proportion to increasing input titers of virus. Imaging could detect infection with three different strains of HSV-1 with the following relative rank order of bioluminescence produced at the site of infection: McKrae > 17 > KOS. Corneal infection with as few as 1 x 10(3) pfu strain McKrae was detectable above background levels. By comparison, infection with vaccinia virus did not affect bioluminescence in the reporter mouse. Collectively, these data establish a new transgenic reporter mouse for infection with HSV-1, thereby enabling in vivo bioluminescence imaging studies of HSV-1 pathogenesis without constructing new reporter viruses.     

Variability of the glycoprotein G gene in clinical isolates of herpes simplex virus type 1.

Glycoprotein G (gG) of herpes simplex virus type 1 (HSV-1) has been used as a prototype antigen for HSV-1 type-specific serodiagnosis, but data on the sequence variability of the gene coding for this protein in wild-type strains are lacking. In this study, direct DNA sequencing of the gG-1 genes from PCR products was performed with clinical HSV-1 isolates from 11 subjects as well as with strains Syn 17(+), F, and KOS 321. The reference strains Syn 17(+) and F showed a high degree of conservation, while KOS 321 carried 13 missense mutations and, in addition, 12 silent mutations. Three clinical isolates showed mutations leading to amino acid alterations: one had a mutation of K(122) to N, which is a gG-1-to-gG-2 alteration; another contained all mutations which were observed in KOS 321 except two silent mutations; and the third isolate carried five missense mutations. Two clinical isolates as well as strain KOS 321 showed a mutation (F(111)-->V) within the epitope of a gG-1-reactive monoclonal antibody (MAb). When all viruses were tested for reactivity with the anti-gG-1 MAb, the three strains with the F(111)-->V mutation were found to be unreactive. Furthermore, gG-1 antibodies purified from sera from the two patients carrying strains mutated in this epitope were less reactive when they were tested by an HSV-1-infected-cell assay. Therefore, our finding that the sequence variability of the gG-1 gene also affects B-cell epitope regions of this protein in clinical isolates may have consequences for the use of this protein as a type-specific antigen for serodiagnosis.     

Herpes simplex virus type 1 recombinants without the oriL sequence replicate DNA with increased fidelity

Herpes simplex virus type 1 (HSV-1) contains three DNA replication origins (ori) of two types. The oriL is located in the center of the unique long sequences, whereas two copies of oriS, which are structurally different from oriL, are within the reiterated sequences flanking the unique short sequences. Recombinant viruses were constructed from ts+7, which contains a deletion of oriL sequences, to have either the beta-galactosidase gene or the supF amplicon integrated into the thymidine kinase locus. Rescue recombinants also were constructed from the supF-containing recombinant to restore the deleted oriL to the wild type sequences. These recombinants were subjected to mutagenesis assays. Results demonstrated that ts+7 viruses with the deletion in oriL sequences replicated both target genes with higher fidelity compared to those derived from the parental strain KOS. Possible mechanisms leading to the high fidelity of DNA replication mediated by viruses without intact oriL sequences are discussed.     

Cloning of herpesviral genomes as bacterial artificial chromosomes

Herpesviruses, which are important pathogens for both animals and humans, have large and complex genomes with a coding capacity for up to 225 open reading frames (ORFs). Due to the large genome size and the slow replication kinetics in vitro of some herpesviruses, mutagenesis of viral genes in the context of the viral genome by conventional recombination methods in cell culture has been difficult. Given that mutagenesis of viral genes is the basic strategy to investigate function, many of the herpesvirus ORFs could not be defined functionally. Recently, a completely new approach for the construction of herpesvirus mutants has been developed, based on cloning of the virus genome as a bacterial artificial chromosome (BAC) in E. coli. This technique allows the maintenance of viral genomes as a plasmid in E. coli and the reconstitution of viral progeny by transfection of the BAC plasmid into eukaryotic cells. Any genetic modification of the viral genome in E. coli using prokaryotic recombination proteins is possible, thereby allowing the generation of mutant viruses and facilitating the analysis of herpesvirus genomes cloned as infectious BACs. In this review, we describe the principle of cloning a viral genome as a BAC using murine gammaherpesvirus 68 (MHV-68), a mouse model for gammaherpesvirus infections, as an example.     

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.     

Variability of the Glycoprotein G Gene in Clinical Isolates of Herpes Simplex Virus Type 1

Glycoprotein G (gG) of herpes simplex virus type 1 (HSV-1) has been used as a prototype antigen for HSV-1 type-specific serodiagnosis, but data on the sequence variability of the gene coding for this protein in wild-type strains are lacking. In this study, direct DNA sequencing of the gG-1 genes from PCR products was performed with clinical HSV-1 isolates from 11 subjects as well as with strains Syn 17⁺, F, and KOS 321. The reference strains Syn 17⁺ and F showed a high degree of conservation, while KOS 321 carried 13 missense mutations and, in addition, 12 silent mutations. Three clinical isolates showed mutations leading to amino acid alterations: one had a mutation of K₁₂₂ to N, which is a gG-1–to–gG-2 alteration; another contained all mutations which were observed in KOS 321 except two silent mutations; and the third isolate carried five missense mutations. Two clinical isolates as well as strain KOS 321 showed a mutation (F₁₁₁→V) within the epitope of a gG-1-reactive monoclonal antibody (MAb). When all viruses were tested for reactivity with the anti-gG-1 MAb, the three strains with the F₁₁₁→V mutation were found to be unreactive. Furthermore, gG-1 antibodies purified from sera from the two patients carrying strains mutated in this epitope were less reactive when they were tested by an HSV-1-infected-cell assay. Therefore, our finding that the sequence variability of the gG-1 gene also affects B-cell epitope regions of this protein in clinical isolates may have consequences for the use of this protein as a type-specific antigen for serodiagnosis.     

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.     

Host species and strain differences affect the ability of an HSV-1 ICP0 deletion mutant to establish latency and spontaneously reactivate in vivo

HSV-1 latency and reactivation were studied in vivo by spontaneous and iontophoresis-induced ocular viral shedding in New Zealand rabbits, Balb/c and A/J mice latently infected with wild-type KOS, and dl x 3.1, a progeny ICP0 deletion mutant. The presence of trigeminal ganglionic latency was demonstrated by the in vitro methods of cocultivation and in situ hybridization. Although the efficiency of ganglionic latency was significantly less (P less than .0001) for dl x 3.1 than for KOS in both mice and rabbits, only dl x 3.1 shed spontaneously in the NZ rabbit. Iontophoresis of adrenergic agents failed to induce reactivation and ocular viral shedding of KOS or dl x 3.1 in mice or rabbits. The establishment of latency and reactivation of KOS and dl x 3.1 was dependent on the host animal. We conclude that host factors as exemplified by host species and host strain differences significantly affected the ability of KOS and dl x 3.1 to establish latency, to reactivate, and to shed spontaneously. ICP0 expression was not required for the establishment or maintenance of latency, nor was it required for the reactivation of latent HSV-1. Furthermore, the biological activity of KOS and dl x 3.1 during latency in vivo did not correlate with latency studies based on in vitro methods.     

Evidence for an involvement of thymidine kinase in the excision repair of ultraviolet-irradiated herpes simplex virus in human cells

A wild-type strain of herpes simplex virus type 1 (HSV-1:KOS) encoding a functional thymidine kinase (tk+) and a tk- mutant strain (HSV-1:PTK3B) were used to study the role of the viral tk in the repair of UV-irradiated HSV-1 in human cells. UV survival of HSV-1:PTK3B was substantially reduced compared with that of HSV-1:KOS when infecting normal human cells. In contrast, the UV survival of HSV-1:PTK3B was similar to that of HSV-1:KOS when infecting excision repair-deficient cells from a xeroderma pigmentosum patient from complementation group A. These results suggest that the repair of UV-irradiated HSV-1 in human cells depends, in part at least, on expression of the viral tk and that the repair process influenced by tk activity is excision repair or a process dependent on excision repair.     

Mutagenesis of murine cytomegalovirus using a Tn3-based transposon

A transposon derived from Escherichia coli Tn3 was introduced into the genome of murine cytomegalovirus (MCMV) to generate a pool of viral mutants. We analyzed three of the constructed recombinant viruses that contained the transposon within the M25, M27, and m155 open reading frames. Our studies provide the first direct evidence to suggest that M25 and M27 are not essential for viral replication in mouse NIH 3T3 cells. Studies in cultured cells and Balb/c mice indicated that the transposon insertion is stable during viral propagation both in vitro and in vivo. Moreover the virus that contained the insertion mutation in M25 exhibited a titer similar to that of the wild-type virus in the salivary glands, lungs, livers, spleens, and kidneys of the Balb/c mice that were intraperitoneally infected with these viruses. These results suggest that M25 is dispensable for viral growth in these organs and the presence of the transposon sequence in the viral genome does not significantly affect viral replication in vivo. The Tn3-based system can be used as a mutagenesis approach for studying the function of MCMV genes in both tissue culture and in animals.     

Marek's disease virus research in the post-sequencing era: new tools for the study of gene functions and virus-host interactions.

Despite the fact that the causative agent of Marek's disease was described more than 30 years ago, and that subsequently many classical biological studies have been carried out on the Marek's disease virus (MDV), detailed analysis of its gene functions has been hampered by lack of suitable research tools. Information on the primary structure of MDV-1 and its serologically related viruses, MDV-2 and herpesvirus of turkeys, is now available. This review focuses on the introduction of the modern and highly efficient technology of bacterial artificial chromosome (BAC) cloning and mutagenesis for rapid manipulation of the MDV genome, with the aim of studying the functions of its genes and non-coding regions. Constructed MDV BACs carry the complete genome of MDV that can be multiplied in Escherichia coli and manipulated using the tools provided by bacterial genetics. The novel approach of MDV DNA mutagenesis using BAC technology will be explained by examples, and we will discuss gene functions in comparison with their counterparts in other herpesviruses. In addition, we have shown that MDV BAC DNA can be used as a polynucleotide vaccine to protect against Marek's disease, thus opening a new chapter in strategies for control of this disease.     

Herpes simplex virus type 1 replicates in the lens and induces cataracts in mice.

In a mouse model, cataracts were produced after corneal inoculation with herpes simplex virus type 1 (HSV-1) strain F. Viral antigen was detected in vivo by the immunoperoxidase technique in lens fibers and occasionally in lens epithelial cell nuclei at 4 and 5 days postinoculation. HSV-1 replication within the lens was further demonstrated by virus isolation from lenses that had been inoculated in vitro. Lens lesions in mice were present as early as 4 days postinoculation and by later times (23 to 60 days postinoculation), severe degenerative changes typical of cataracts were present in many of the lenses examined histologically. Viral DNA was detected in extracts of lenses from infected animals at 5 and 11 days postinoculation. In contrast to HSV-1 F, the KOS strain of HSV-1 produced intraocular infection and cataracts at a much lower frequency in mice. Lens lesions appear to be induced directly by viral replication within the lens; however, other mechanisms involving inflammation in adjacent eye structures may also be involved in the pathogenesis of these lesions. Based upon these data, it is reasonable to consider the possibility that HSV-1 may be involved in the production of cataracts in humans in some cases.     

Rapid identification of non-essential genes for in vitro replication of Marek's disease virus by random transposon mutagenesis

Marek's disease virus (MDV) is a highly oncogenic alphaherpesvirus that induces rapid-onset T-cell lymphomas in poultry. The MDV genome encodes more than 100 genes. However, the role of many of these genes in virus replication is not known. The construction of an infectious bacterial artificial chromosome (BAC) clone of the highly oncogenic RB-1B strain of MDV has been described previously. Virus reconstituted from the BAC clone induced rapid-onset lymphomas in chickens very similar to the wildtype viruses. In this paper, the construction of a high-density random transposon-insertion mutant library of the RB-1B BAC clone using a high throughput in vitro transposon mutagenesis technique is described. Furthermore a PCR screening method, using primers specific for the transposon sequence and the MDV gene(s) of interest, was developed for the rapid identification of specific insertion mutants. The application of the screening method to identify some of the non-essential genes for MDV replication in vitro is described.