JVI Figure table search 04
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Blakeney, S.
Right arrow Articles by Visalli, R. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Blakeney, S.
Right arrow Articles by Visalli, R. J.

 Previous Article  |  Next Article 

Journal of Virology, August 2005, p. 10498-10506, Vol. 79, No. 16
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.16.10498-10506.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Herpes Simplex Virus Type 2 UL24 Gene Is a Virulence Determinant in Murine and Guinea Pig Disease Models

Susan Blakeney,1 Jacek Kowalski,1 Donna Tummolo,1 Joanne DeStefano,1 David Cooper,1 Min Guo,1 Seema Gangolli,1 Deborah Long,1 Timothy Zamb,1 Robert J. Natuk,1 and Robert J. Visalli1,2*

Wyeth Vaccines Research, Pearl River, New York 10965,1 Department of Biology, Indiana University-Purdue University Fort Wayne, Fort Wayne, Indiana 468052

Received 25 March 2005/ Accepted 4 May 2005


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A herpes simplex virus type 2 (HSV-2) UL24 ß-glucuronidase (UL24-ßgluc) insertion mutant was derived from HSV-2 strain 186 via standard marker transfer techniques. Cell monolayers infected with UL24-ßgluc yielded cytopathic effect with syncytium formation. UL24-ßgluc replicated to wild-type viral titers in three different cell lines. UL24-ßgluc was not virulent after intravaginal inoculation of BALB/c mice in that all inoculated animals survived doses up to 400 times the 50% lethal dose (LD50) of the parental virus. Furthermore, few UL24-ßgluc-inoculated mice developed any vaginal lesions. Intravaginal inoculation of guinea pigs with UL24-ßgluc at a dose equivalent to the LD50 of parental virus ({approx}5 x 103 PFU) was not lethal (10/10 animals survived). Although genital lesions developed in some UL24-ßgluc-inoculated guinea pigs, both the overall number of lesions and the severity of disease were far less than that observed for animals infected with parental strain 186.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Understanding of the role of herpes simplex virus type 2 (HSV-2) genes and their corresponding gene products in appropriate models of viral pathogenesis has lagged behind similar studies performed on HSV-1 counterparts. The vaginal murine and guinea pig models of herpetic disease provide an opportunity to study the contribution of HSV-2 gene products during both the acute and latent phases of disease. In particular, the guinea pig model mimics that of human disease in that one can observe acute disease and subsequent reactivation from latency. Despite such a model, few HSV-2 genes have been characterized for their role in genital disease. Understanding which gene products are important for attenuation at the level of acute and latent disease is necessary if one is to design an effective live, attenuated vaccine.

To date, studies using the guinea pig model have characterized the role of only a few HSV-2 loci, the viral thymidine kinase (TK) (1), the large subunit of ribonucleotide reductase (27), and the latency-associated transcript region (11, 28, 31). Studies of HSV-2 mutants in murine models have included the characterization of the HSV-2 TK (15), US2 and US3 (6), virion host shut-off (19), and glycoprotein C (9) genes. In this report, we characterize the role the HSV-2 UL24 gene product in both murine and guinea pig vaginal models of disease.

The HSV-2 UL24 gene product was recently shown to be a 32-kDa virion structural protein (5), but currently no information is available describing the role of HSV-2 UL24 during in vitro infection or in viral pathogenesis in vivo. DNA sequence analysis has revealed that UL24 homologs are present throughout the Herpesviridae family. The herpes simplex virus type 1 (HSV-1) UL24 gene product was shown to be a 30-kDa nucleus-associated protein (16) that is not required for growth in cultured cells (7). The UL24 homolog identified in bovine herpesvirus type 1 (BHV-1) was shown to have a transcription profile similar to that of HSV-1 UL24. Deletion of the BHV-1 UL24 open reading frame (ORF) had little effect on viral replication in vitro (30).

Although the function(s) of the UL24 protein is not known, mutation of the HSV-1 gene results in the development of a syncytial plaque-forming phenotype following infection of certain cell types in vitro (7, 8, 23). Thus, the UL24 protein may regulate fusogenic activity during HSV infection. There are a number of different HSV gene products whose alteration or mutation appears to affect fusogenic activity during infection. The in vivo significance of gene products that modulate fusion is not yet understood. Studies using HSV-1 UL24 point mutants in a murine ocular disease model suggested that the HSV-1 UL24 gene product was important for peripheral replication in corneal tissue, acute replication in sensory ganglia, and reactivation from explanted mouse ganglia (7).

We are interested in studying the contribution of HSV-2 gene products to viral replication and virulence. In this study, a UL24 ß-glucuronidase (UL24-ßgluc) insertion mutant and the corresponding repaired virus (UL24R) were constructed and characterized for the ability to (i) replicate in three different cell lines in vitro and (ii) cause disease after intravaginal inoculation in both murine and guinea pig disease models.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Viruses and cells. African green monkey kidney (Vero), human foreskin fibroblast (HFF), and SK-N-SH neuroblastoma cell lines were grown in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum, 100 units/ml of penicillin, and 100 µg/ml of streptomycin sulfate. Cells infected with virus were grown in Dulbecco's modified Eagle's medium containing 2% serum and antibiotics.

HSV-2 strain 186 was propagated and titered on Vero cells and used as the parental virus for these studies.

Construction and isolation of UL24-Bgluc and UL24R. The region containing and flanking the UL24 gene (Fig. 1, speckled box) was amplified by PCR from wild-type HSV-2 strain 186 DNA and cloned into the PCR-Blunt II-TOPO vector (Invitrogen, Carlsbad, CA). The region containing the HSV-2 186 UL23 and UL24 genes was sequenced and compared to that of HSV-2 HG52 and HSV-1 17 using Clustal W alignment.



View larger version (17K):
[in this window]
[in a new window]
  		FIG. 1. Structure of the HSV-2 genome and the region encoding the UL24 gene. The diagram demonstrates the locations of the UL23, 24, and 25 ORFs and the direction of transcription (arrows) for the parental strain, HSV-2 186 (3). Details of HSV-2 UL24 gene transcription are not known, but the mapping of transcripts in the analogous region of HSV-1 has been summarized previously (2, 17). A restriction map of the UL24 gene and the adjoining regions is provided. A ß-glucuronidase marker cassette was inserted at the BglII site within the UL24 ORF. A restriction map of the ß-glucuronidase marker cassette and adjoining regions is provided to indicate the predicted structure of UL24-ßgluc. The numbers in parentheses represent the nucleotide positions based on the sequence of HSV-2 (3) and were renumbered for UL24-ßgluc after the insertion of the 2.8-kb ß-glucuronidase cassette. The speckled box represents the region of the genome subcloned for manipulation of the UL24 gene. Sequence analysis confirmed the presence of restriction sites also found in HSV-2 HG52 (underlined). This fragment contained the entire UL24 ORF and portions of the UL23 and UL25 ORFs. In UL24-ßgluc, the hatched box represents the coding sequences of the inserted ß-glucuronidase gene. The arrow below the box indicates the predicted ß-glucuronidase transcript. The small open box and the cross-hatched box represent the simian virus 40 polyadenylation and promoter sequences of the ß-glucuronidase cassette, respectively. The probes utilized in Southern blot analysis are indicated.

 
UL24-ßgluc was constructed by inserting a ß-glucuronidase cassette (Clonetech, Palo Alto, CA) into the conserved BglII site of the HSV-2 186 UL24 ORF (Fig. 1). Briefly, the plasmid containing the UL24 gene was digested at the single BglII site and a ß-glucuronidase cassette with BamHI ends was inserted as depicted in Fig. 1. The ß-glucuronidase cassette (approximately 2.8 kb) comprises the ß-glucuronidase gene (approximately 1.9 kb), simian virus 40 promoter and simian virus 40 polyadenylation sequences. The plasmid containing the disrupted UL24 gene was linearized and transfected into Vero cells that were subsequently infected with HSV-2 186 (25). Transfection/infection stocks were plated in the presence of the chromogenic substrate 5-bromo-4-chloro-3-indolyl-ß-D-glucuronide (X-Gluc) and blue plaque mutants were selected from the background of white parental 186 viruses as previously described (10). Blue plaques representing viral recombinants were plaque purified three times, and one plaque, UL24-ßgluc, was selected for further study.

The unaltered, TOPO-cloned DNA fragment containing the HSV-2 186 UL24 gene was used to repair UL24-ßgluc. This fragment was transfected into Vero cells that were subsequently infected with UL24-ßgluc. White, nonsyncytial plaques were purified and a single UL24-repaired virus, UL24R, was selected for further study.

Southern blot analysis. Viral DNA was isolated from partially purified virions, digested with restriction enzymes (BamHI, NcoI, and SacI), and electrophoresed through agarose gels. The DNAs were blotted to positively charged nylon membranes and hybridized to either a 600-bp HSV-2 fragment (UL24 5' probe) or ß-glucuronidase-specific sequences (ß-glucuronidase probe) (Fig. 1). Double-stranded DNA probes were radiolabeled with [{alpha}-33P]dCTP and viral DNA fragments hybridizing to the probes were detected by autoradiography.

In vitro viral replication and viral plaque morphology. Three cell types, Vero, HFF, and SK-N-SH, were infected with either HSV-2 186, UL24-ßgluc, or UL24R at a multiplicity of infection (MOI) of 0.01 or 5.0. Photographs were taken to document cytopathic effect. Infected cells were harvested at the indicated time points, subjected to three cycles of freeze-thawing, processed in a cup-horn sonicator for approximately 15 s, and clarified by low-speed centrifugation (2,000 x g). A commercial enzyme-linked virus-inducible system (20) (ELVIS; Diagnostic Hybrids, Inc., Athens, OH) in a 96-well format was used to estimate the titer of all growth kinetics samples. Using this system, quantitation of viral titers is typically performed by counting blue cell-forming units (BFU). Previous studies revealed that the number of BFU/ml closely approximates the number of PFU/ml as determined by plaque assay (20). Since we have consistently observed a positive correlation between ß-galactosidase levels, BFU, and PFU, a soluble ß-galactosidase assay was employed to estimate the amount of virus present in each sample (14).

In brief, infected cell samples and a positive control sample containing a known amount of virus were serially diluted in serum-free medium and incubated for 1 hour at 37°C, after which 75 µl was transferred to a well of the ELVIS plate containing 75 µl of serum-free medium. Following a second 1 hour of incubation at 37°C, 75 µl of ELVIS replacement medium was added to each well and plates were placed in a 37°C CO2 incubator for 16 to 18 h. Medium was replaced with 50 µl of 1% Triton X-100, and plates were frozen at –70°C for a minimum of 2 h. Plates were thawed and 50 µl of 2X chlorophenol red-ß-D-galactopyranoside (CPRG, 16 mM), 0.12 M Na2HPO4, 0.08 M NaH2PO4, 0.02 M KCl, 2 mM MgSO4 and 0.01 M ß-mercaptoethanol, pH 7.8, substrate was added followed by 1 to 3 h of incubation at 37°C. Optical density (OD) values for the various sample dilutions were determined at 570 nm. Values obtained for the infected cell samples were utilized to estimate viral titers by comparison with a standard curve generated from the positive control sample results.

Plaque reduction assay. Plaque reduction assays were performed as previously described (26) with the following modifications. Vero cells were infected with approximately 50 to 100 PFU of virus per well. Acyclovir was diluted to the desired concentrations in Dulbecco's modified Eagle's medium and applied to uninfected Vero cell monolayers for a 30-minute preincubation prior to the addition of virus. Positive control wells received virus without acyclovir. Monolayers were incubated for 3 days at 37°C, fixed, and counted. Data are presented as the mean of at least three independent assays.

Murine vaginal model. Eight-week-old female BALB/c mice were obtained from Taconic Laboratories Animals and Services (Germantown, NY). Mice were housed in microisolator cages (five animals/cage) and were permitted to feed and drink ad libitum. Transponders (BioMedic Data Systems Inc., Rockville, MD) were inserted subcutaneously into the backs of mice as per the manufacturer's instructions. Using a DAS-5001 Desktop scanner linked to a Sartorius balance (Sartorius Corporation, Edgewood, NY), transponders were used to identify mice and take and record body weights and temperatures. All animal protocols employed in this study met with established Institutional Animal Care and Use Committee guidelines.

Five days prior to virus challenge, all mice received 2.0 mg Depo-Provera (Upjohn Company, Kalamazoo, MI) subcutaneously in the scruff of the neck to synchronize their estrous cycles and to increase their susceptibility to HSV-2 vaginal infection (15). For infection, mice were anesthetized and the vaginas were swabbed with a sterile Dacron polyester tip applicator (Puritan, Guilford, ME) prewet in phosphate-buffered saline to remove any associated mucus. Mice were subsequently inoculated intravaginally with the indicated doses of either wild-type HSV-2 strain 186, UL24-ßgluc, or UL24R. Virus was instilled into the vaginal vault with the aid of a micropipettor (0.01 ml/dose). The mice were monitored daily for 4 weeks for symptoms of viral infection and mortality.

Mice were scored for signs of disease using the following scale: 0, no symptoms; 1, vaginal erythema; 2, vaginal erythema and edema; 3, vaginal herpetic lesions; 4, unilateral paralysis; and 5, bilateral paralysis or death. The mean severity index was determined by taking the mean score of all mice within a group. All mice that were bilaterally paralyzed or showed signs of severe illness and/or distress (fever or weight loss) were immediately subjected to euthanasia.

Guinea pig model of herpetic disease. Hartley albino out-bred female guinea pigs (250 to 350 g) were obtained from Charles River Laboratories (Kingston, N.Y.). Animals were prepared for intravaginal inoculation by first swabbing the region with a calcium alginate swab prewet in phosphate-buffered saline, followed by swabbing with a dry swab to remove vaginal mucus that might interfere with virus uptake. The desired dose of virus was formulated in 100 µl of phosphate-buffered saline and slowly instilled into the vaginal vault with a 1-ml syringe fitted with 0.5 inch (ca. 1.5 cm) of narrow butterfly tubing.

Disease scoring was performed as previously described by the method of Stanberry et al. (21). Acute disease was scored between days 3 and 10 postinoculation. The mean lesion score represents the average disease score of all surviving animals on that day of scoring. Recurrent disease was scored by counting lesions each day between days 15 and 56 postinoculation. The average lesions per animal in the group were expressed cumulatively over the noted time period. Animals were constantly observed for signs of severe disease, including paralysis and central nervous system involvement. Mortality values reflect animals that were sacrificed due to distress.

Detection of HSV-2 DNA in guinea pig dorsal root ganglia. Sacral dorsal root ganglia (six per animal) were dissected at the termination of the experiment and weighed and the viral DNA was extracted using a QIAamp DNA mini kit (QIAGEN). Real-time PCR was performed on extracted DNA samples. A standard curve was constructed for each experiment using purified plasmid containing HSV-2 glycoprotein D gene sequences. Data were normalized using probes specific for guinea pig lactalbumin DNA in order to correct for variable amounts of neural material in the dissected ganglia.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Construction and isolation of UL24-ßgluc and UL24R. The UL24-ßgluc insertion mutant was isolated as described in the Materials and Methods. Based on the predicted amino acid sequence of HSV-2 UL24 (281 amino acids), insertion into the BglII site of the UL24 ORF (Fig. 1) should result in a truncated UL24 polypeptide lacking the final 100 C-terminal amino acid residues (i.e., Ser 182 to Glu 281) of the full-length UL24 protein. The insertion mutation was designed so that the overlapping UL23 TK gene transcript would not be disrupted but, conversely, all six potential UL24 transcripts (shown to be expressed from the HSV-1 UL24 gene) (2) would be disrupted.

The nucleotide sequence of HSV-2 186 in the region containing the 5'-overlapping UL23 and UL24 ORFs was sequenced and compared to that of HSV-1 17 and HSV-2 HG52 via Clustal W alignment (Fig. 2). The 186 sequence was nearly identical to that of HG52 and had high identity to HSV-1 17. Although we have not mapped the transcripts for HSV-2 186 UL24, the genetic layout and sequence relatedness suggest that expression of HSV-2 186 UL24 would not be significantly different from that of HSV-1. Furthermore, for this study, we constructed a single insertion mutation clearly within the UL24 ORF, and no attempt is made to dissect a role for any particular UL24 transcript.



View larger version (44K):
[in this window]
[in a new window]
  		FIG. 2. Comparison of HSV-2 186, HSV-2 HG52, and HSV-1 17 sequences by Clustal W alignment. Asterisks represent identity among all three viruses. The gray box shows the only observed difference between 186 and HG52 in the region represented. The ATG for the UL24 gene is underlined, and the direction of transcription is indicated by an arrow. The position of the ATG for the UL23 (TK) gene is identical for the three viruses. The direction of UL23 transcription is opposite that of UL24 and is represented by a dashed arrow.

 
A cloned DNA fragment containing the HSV-2 186 UL24 gene was used to repair UL24-ßgluc and generate UL24R.

Analysis of recombinant viral genomes. Figure 1 is a schematic representation of the genomic structure for the region encoding the UL24 gene. Restriction maps are provided for parental HSV-2 strain 186 and UL24-ßgluc. The locations of two DNA probes utilized in Southern analysis are indicated (Fig. 1; ß-gluc and UL24 5' probes).

Viral DNAs were analyzed by Southern blotting (Fig. 3) to confirm that they had the expected genomic structures. HSV-2 186, UL24-ßgluc, and UL24R DNAs were digested with BamHI, NcoI, or SacI and probed with a 600-base-pair fragment containing 3' UL23 and 5' UL24 sequences (UL24 5' probe, Fig. 1). Based on the restriction map in Fig. 1, HSV-2 186 and UL24R digested DNAs should yield fragments of 3.3 and 4.1 kb, 4.5 kb, and 4.4 kb after digestion with BamHI, NcoI, and SacI, respectively. Insertion of the ß-glucuronidase cassette into the BglII site of the UL24 gene would introduce new restriction sites resulting in fragments of 3.3 and 6.7 kb, 4.0 kb, and 6.9 kb after digestion with BamHI, NcoI, and SacI, respectively. The predicted hybridization patterns were observed for all three viruses.



View larger version (83K):
[in this window]
[in a new window]
  		FIG. 3. Southern blot analysis. Viral DNAs were digested with restriction enzyme BamHI (B), NcoI (N), or SacI (S), electrophoresed through agarose, blotted to nylon membrane, and hybridized to a 600-bp HSV-2 fragment (UL24 5' probe). Molecular size markers (kb) are provided.

 
Using ß-glucuronidase DNA sequences as a probe (Fig. 1, ß-gluc probe), UL24-ßgluc DNA fragments of 6.7 kb (BamHI), 4.0 kb, 2.8 kb, and 0.1 kb (NcoI), and 6.9 kb (SacI) were observed, as predicted (data not shown). As expected, the ß-glucuronidase probe did not hybridize with any 186 or UL24R DNA fragments.

In vitro cytopathic effect and in vitro replication. The cytopathic effect after infection of three different cell types with either 186, UL24-ßgluc, or UL24R was examined. Vero, HFF, and SK-N-SH cell monolayers displayed extensive syncytium formation after infection with UL24-ßgluc (Fig. 4). Nonsyncytial plaques were not observed. No syncytia formed after infection with either 186 or UL24R.



View larger version (107K):
[in this window]
[in a new window]
  		FIG. 4. Cytopathic effect. Three different cell types were infected with the indicated viruses. Approximately 48 h postinfection, a representative field of each monolayer was photographed at an identical magnification.

 
The ability of the viruses to replicate in vitro was analyzed. Vero, HFF, or SK-N-SH cells were infected at either low (0.01) or high (5.0) MOI. Figure 5a shows the total virus yield obtained at 18 h postinfection with an MOI of 5.0. All three viruses replicated to similar titers ({approx}106 PFU) in each of the cell types.



View larger version (25K):
[in this window]
[in a new window]
  		FIG. 5. Replication of viruses in vitro. Monolayers of Vero (African green monkey kidney), HFF (human foreskin fibroblast), or SK-N-SH (neuroblastoma) cells were infected at either a low (0.01) or high (5.0) MOI. Monolayers were washed 1 h after infection and overlaid with fresh growth medium. Infected cell monolayers were harvested at 18 h for the high-MOI infections and at 24, 30, 36, and 48 h for the low-MOI infections. Samples were frozen and thawed three times, briefly sonicated, and then cleared via low-speed centrifugation. Duplicate samples were prepared for the Vero and HFF infections. Panel a shows the total virus yield obtained at 18 h postinfection for each of the viruses in the three cell types. Panels b, c, and d indicate viral replication and spread in Vero, HFF, and SK-N-SH cells, respectively, from 24 to 48 h after low-MOI infection.

 
Low MOI (0.01) was utilized to examine the ability of the three viruses to replicate and spread in the three cell types (Fig. 5b, c, and d). Regardless of the cell type employed, all three viruses replicated and spread with comparable kinetics, as indicated by the increasing titers.

The results shown in Fig. 5 indicated that insertion of the ß-glucuronidase cassette into the BglII site of the UL24 gene did not drastically affect the ability of the virus to replicate in vitro. Combined with the plaque morphology data, these results suggest that the C-terminal one-third of the UL24 gene product was important, either directly or indirectly, in modulating fusion events in HSV-2-infected cells but that the full-length UL24 gene product was not required for replication in the cell types tested.

TK function (sensitivity to acyclovir). The proximity of the UL24 and UL23 (TK) genes created the possibility that mutation of the UL24 gene could affect the expression of the TK gene (Fig. 2). This presented a major concern in previous studies when trying to determine the separate roles of the TK or UL24 genes in viral replication, particularly in viral pathogenesis (4, 8, 12, 13, 18). The insertion mutation within UL24-ßgluc was such that it should not have any deleterious effects on the expression and therefore the function of the HSV-2 TK gene. The lack of an effect on HSV-2 TK function was demonstrated by examination of the sensitivity of the three viruses to increasing concentrations of acyclovir (Fig. 6). All three viruses showed a similar 50% inhibitory concentration of approximately 3 µM. Hence, this result strongly suggested that the TK activity (phosphorylation of acyclovir) was similar for all three viruses and that any properties or phenotypes observed for UL24-ßgluc are independent of TK activity.



View larger version (13K):
[in this window]
[in a new window]
  		FIG. 6. Viral plaque reduction assay to test sensitivity to acyclovir. The viruses were plated on Vero cell monolayers in the presence of various concentrations (0 to 16 µM) of acyclovir. Plaques that formed 72 h after infection were counted and the data were used to generate 50% inhibitory concentration (IC50) values for each virus.

 
Pathogenesis in mice. A murine intravaginal infection model was employed to evaluate the ability of UL24-ßgluc to cause morbidity and mortality in vivo (Fig. 7). Survival curves (Fig. 7a) indicated that HSV-2 186 killed 100% of mice infected with either 250 or 1.25 x 104 PFU and UL24R killed 70% and 100% at 250 and 1.25 x 104 PFU, respectively. In contrast, a total of 80 animals (10 per group) were inoculated with various doses of UL24-ßgluc, ranging from 250 to 105 PFU, and all of the animals survived during the 4-week observation period following viral inoculation.





View larger version (72K):
[in this window]
[in a new window]
  		FIG. 7. In vivo mouse data, including (a) mortality curves, (b) lesion scores, and (c) disease progression. Mice were anesthetized, vaginas were swabbed, and the indicated dose (PFU) of each virus was gently instilled into the vaginal vaults with the aid of a micropipettor. Scoring: 0, no symptoms; 1, vaginal erythema; 2, vaginal erythema and edema; 3, vaginal herpetic lesions; 4, unilateral paralysis; and 5, bilateral paralysis or death. The mean severity index was determined by taking the mean score of all mice within a group (error bars represent the standard deviation of the mean).

 
Similar results were observed when measuring lesion formation or disease progression (based on severity score; see Materials and Methods). Lesion formation was observed in mice infected with either 186 or UL24R at either dose (Fig. 7b). One hundred percent of the infected animals showed herpetic lesions for both 186 groups and the high-dose UL24R group by day 10. Eighty percent of the low-dose UL24R group presented with lesions. In contrast, very few of the 80 UL24-ßgluc-inoculated animals presented with lesion formation. A small amount of lesion formation was scored at day 10 in two of the eight UL24-ßgluc groups.

Disease severity scores mimicked lesion formation (Fig. 7c). The severity of disease was delayed and reduced in all of the UL24-ßgluc-infected mice, where average disease scores ranged from no symptoms to mild vaginal erythema.

Pathogenesis in guinea pigs. The guinea pig intravaginal model for HSV-2 is well established and has been shown to mimic both the acute and latent phases of human herpetic disease (21). Since HSV-2 186 was shown to have a relatively low LD50 in guinea pigs, we decided to perform the guinea pig experiments with an inoculum that was approximately the LD50 of strain 186. The mortality data showed that HSV-2 186 killed 80% of the guinea pigs at a dose of 3 x 103 PFU by day 30, whereas UL24-ßgluc, administered at a similar dose, did not kill any animals (Fig. 8a). We observed the guinea pigs for symptoms of acute disease during the first 8 days after intravaginal inoculation (before the 186-infected animals began to die). Acute disease (Fig. 8b) was generally higher (mean lesion score, {approx}2.1) for 186-infected animals, whereas UL24-ßgluc-infected animals showed only low-level indications of infection (mean lesion score, {approx}0.5).



View larger version (10K):
[in this window]
[in a new window]
  		FIG.8. In vivo guinea pig data, including (a) mortality curves, (b) acute disease scores, and (c) reactivation scores. Hartley guinea pigs were inoculated with 100 µl of HSV-2 in the vaginal vault. Scoring was performed by the method of Stanberry et al. (21).

 
The UL24-ßgluc-infected animals were followed from days 15 to 50 postinfection for signs of reactivation from latency (Fig. 8c). The appearance of lesions in some animals was indicative that the UL24-ßgluc mutant could establish latency in the dorsal root ganglia of intravaginally infected guinea pigs and that the mutant virus could reactivate to form herpetic lesions. Furthermore, real-time quantitative PCR performed on ganglia removed on day 50 postinfection from the 10 UL24-ßgluc animals indicated that at least 7/10 (70%) of the UL24-ßgluc-infected guinea pigs contained detectable HSV-2 DNA in their ganglia. The results suggested that UL24-ßgluc was able to establish a latent infection in guinea pig dorsal root ganglia that could reactivate to produce lesions after the resolution of the acute disease phase. It was not possible to directly compare the levels of reactivation to wild-type virus in this experiment due to the high mortality rate observed with strain 186. Further studies will be required to accurately determine if the number of genome copies per ganglion differs between UL24-ßgluc- and 186-infected animals.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A number of herpesvirus genes have been shown to be nonessential for growth in cultured cells. However, when viral mutants were tested in certain animal models, a number of these genes proved to be important in promoting viral replication and disease in vivo. (reviewed in references 22, 24, and 29). The studies presented here provide new information of the contribution of the UL24 gene in herpes simplex virus pathogenesis. We described the isolation of a UL24 insertion mutant that is viable in vitro yet shows significant attenuation in two different small-animal disease models.

Analysis of the role of the UL24 gene in the viral life cycle in vitro and in vivo has been complicated by the fact that certain mutations in UL24 can affect the expression of the overlapping UL23 (TK) gene (4, 8, 12, 13, 18). We addressed these concerns by measuring the sensitivity of UL24-ßgluc to acyclovir, the substrate for viral TK. The results demonstrate that the ß-glucuronidase cassette, inserted at the BglII site used to disrupt the UL24 ORF, had no obvious effect on the expression or function of the HSV-2 TK as measured by sensitivity to acyclovir.

Our results suggest that the full-length HSV-2 UL24 protein is not required for viral replication in vitro. However, insertion mutagenesis of the UL24 gene resulted in a syncytial plaque phenotype in the three cell types tested. It is likely that the C-terminal one-third of the UL24 gene product is important, either directly or indirectly, in modulating fusion events in the infected cell but that this region was not essential for viral replication in vitro.

The role of the UL24 gene in vivo was assessed by intravaginal inoculation of parental and recombinant viruses into BALB/c mice and Hartley guinea pigs. An HSV-2 UL24 mutant was avirulent in mice at doses up to at least 400 times the parental virus LD50, the highest dose tested. Intravaginal infection of mice with a UL24 mutant resulted in delayed disease kinetics and minimal disease progression, including lesion formation. Low levels of acute herpetic disease with no associated mortality were observed in guinea pigs following intravaginal infection with the UL24 mutant at a dose that was at least equivalent to the LD50 of the parental virus.

Our studies showed that UL24-ßgluc was able to establish latency in the dorsal root ganglia of intravaginally infected guinea pigs and could reactivate to yield characteristic herpetic lesions, albeit at lower levels than those observed for wild-type HSV-2 strains (data not shown). Previously, HSV-1 UL24 mutants examined in a murine ocular virus challenge model suggested a role for UL24 in the establishment and reactivation from latency in mouse ganglia (7).

The similarities and differences between HSV-1 and HSV-2 UL24 mutants with respect to latency are difficult to assess. HSV-1 UL24 mutants were significantly impaired for their ability to replicate in trigeminal ganglia and in their ability to reactivate from latency, as determined by cocultivation and dissociation of ganglia. Similarly, our studies suggested that the UL24-ßgluc was impaired for the ability to replicate and/or spread in the guinea pig central nervous system, since no mortality or morbidity (paralysis) was observed at the dose tested. However, UL24-ßgluc was able to establish latency in guinea pig sacral ganglia in that 7 of 10 animals were positive for HSV-2 DNA sequences 50 days postinoculation. The HSV-2 UL24 mutant could reactivate from latency in the guinea pig model, but both the number of lesions and severity of disease never approached that of other wild-type HSV-2 strains (186 and MS) that we have tested (data not shown).

Further studies of the HSV-2 UL24 protein will be necessary to understand why UL24-ßgluc replication in cell culture was similar to that of wild-type virus yet was clearly attenuated with respect to disease in both the murine and guinea pig models. The guinea pig model should prove valuable in evaluating both the prophylactic and therapeutic potential of UL24 mutants as attenuated vaccine candidates.


   ACKNOWLEDGMENTS
 
We thank Aaron Abramovitz for helpful discussions.


   FOOTNOTES
 
* Corresponding author. Present address: Department of Biology, Indiana University-Purdue University Fort Wayne, 2101 E. Coliseum Blvd., Fort Wayne, IN 46805-1499. Phone: (260) 481-6320. Fax: (260) 481-6087. E-mail: visallir{at}ipfw.edu. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Bernstein, D. I., J. Ireland, and N. Bourne. 2000. Pathogenesis of acyclovir-resistant herpes simplex type 2 isolates in animal models of genital herpes: models for antiviral evaluations. Antiviral Res. 47:159-169.[CrossRef][Medline]
  2. Cook, W. J., and D. M. Coen. 1996. Temporal regulation of herpes simplex virus type 1 UL24 mRNA expression via differential polyadenylation. Virology 218:204-213.[CrossRef][Medline]
  3. Dolan, A., F. E. Jamieson, C. Cunningham, B. C. Barnett, and D. J. McGeoch. 1998. The genome sequence of herpes simplex virus type 2. J. Virol. 72:2010-2021.[Abstract/Free Full Text]
  4. Ho, D. Y., and E. S. Mocarski. 1988. Beta-galactosidase as a marker in the peripheral and neural tissues of the herpes simplex virus-infected mouse. Virology 167:279-283.[CrossRef][Medline]
  5. Hong-Yan, Z., T. Murata, F. Goshima, H. Takakuwa, T. Koshizuka, Y. Yamauchi, and Y. Nishiyama. 2001. Identification and characterization of the UL24 gene product of herpes simplex virus type 2. Virus Genes 22:321-327.[CrossRef][Medline]
  6. Inagaki-Ohara, K., T. Iwasaki, D. Watanabe, T. Kurata, and Y. Nishiyama. 2001. Effect of the deletion of US2 and US3 from herpes simplex virus type 2 on immune responses in the murine vagina following intravaginal infection. Vaccine 20:98-104.[CrossRef][Medline]
  7. Jacobson, J. G., S. H. Chen, W. J. Cook, M. F. Kramer, and D. M. Coen. 1998. Importance of the herpes simplex virus UL24 gene for productive ganglionic infection in mice. Virology 242:161-169.[CrossRef][Medline]
  8. Jacobson, J. G., S. L. Martin, and D. M. Coen. 1989. A conserved open reading frame that overlaps the herpes simplex virus thymidine kinase gene is important for viral growth in cell culture. J. Virol. 63:1839-1843.[Abstract/Free Full Text]
  9. Johnson, D. C., M. R. McDermott, C. Chrisp, and J. C. Glorioso. 1986. Pathogenicity in mice of herpes simplex virus type 2 mutants unable to express glycoprotein C. J. Virol. 58:36-42.[Abstract/Free Full Text]
  10. Jones, T. R., V. P. Muzithras, and Y. Gluzman. 1991. Replacement mutagenesis of the human cytomegalovirus genome: US10 and US11 gene products are nonessential. J. Virol. 65:5860-5872.[Abstract/Free Full Text]
  11. Krause, P. R., L. R. Stanberry, N. Bourne, B. Connelly, J. F. Kurawadwala, A. Patel, and S. E. Straus. 1995. Expression of the herpes simplex virus type 2 latency-associated transcript enhances spontaneous reactivation of genital herpes in latently infected guinea pigs. J. Exp. Med. 181:297-306.[Abstract/Free Full Text]
  12. Leist, T. P., R. M. Sandri-Goldin, and J. G. Stevens. 1989. Latent infections in spinal ganglia with thymidine kinase-deficient herpes simplex virus. J. Virol. 63:4976-4978.[Abstract/Free Full Text]
  13. Meignier, B., R. Longnecker, P. Mavromara-Nazos, A. E. Sears, and B. Roizman. 1988. Virulence of and establishment of latency by genetically engineered deletion mutants of herpes simplex virus 1. Virology 162:251-254.[CrossRef][Medline]
  14. Nussbaum, O., C. C. Broder, and E. A. Berger. 1994. Fusogenic mechanisms of enveloped-virus glycoproteins analyzed by a novel recombinant vaccinia virus-based assay quantitating cell fusion-dependent reporter gene activation. J. Virol. 68:5411-5422.[Abstract/Free Full Text]
  15. Parr, M. B., L. Kepple, M. R. McDermott, M. D. Drew, J. J. Bozzola, and E. L. Parr. 1994. A mouse model for studies of mucosal immunity to vaginal infection by herpes simplex virus type 2. Lab. Investig. 70:369-380.[Medline]
  16. Pearson, A., and D. M. Coen. 2002. Identification, localization, and regulation of expression of the UL24 protein of herpes simplex virus type 1. J. Virol. 76:10821-10828.[Abstract/Free Full Text]
  17. Rajcani, J., V. Andrea, and R. Ingeborg. 2004. Peculiarities of herpes simplex virus (HSV) transcription: an overview. Virus Genes 28:293-310.[Medline]
  18. Sears, A. E., B. Meignier, and B. Roizman. 1985. Establishment of latency in mice by herpes simplex virus 1 recombinants that carry insertions affecting regulation of the thymidine kinase gene. J. Virol. 55:410-416.[Abstract/Free Full Text]
  19. Smith, T. J., L. A. Morrison, and D. A. Leib. 2002. Pathogenesis of herpes simplex virus type 2 virion host shutoff (vhs) mutants. J. Virol. 76:2054-2061.[Abstract/Free Full Text]
  20. Stabell, E. C., and P. D. Olivo. 1992. Isolation of a cell line for rapid and sensitive histochemical assay for the detection of herpes simplex virus. J. Virol. Methods 38:195-204.[CrossRef][Medline]
  21. Stanberry, L. R., E. R. Kern, J. T. Richards, T. M. Abbott, and J. C. Overall, Jr. 1982. Genital herpes in guinea pigs: pathogenesis of the primary infection and description of recurrent disease. J. Infect. Dis. 146:397-404.[Medline]
  22. Subak-Sharpe, J. H., and D. J. Dargan. 1998. HSV molecular biology: general aspects of herpes simplex virus molecular biology. Virus Genes 16:239-251.[CrossRef][Medline]
  23. Tognon, M., R. Guandalini, M. G. Romanelli, R. Manservigi, and B. Trevisani. 1991. Phenotypic and genotypic characterization of locus Syn 5 in herpes simplex virus 1. Virus Res. 18:135-150.[CrossRef][Medline]
  24. Visalli, R. J., and C. R. Brandt. 2002. Mutation of the herpes simplex virus 1 KOS UL45 gene reveals dose dependent effects on central nervous system growth. Arch. Virol. 147:519-532.[CrossRef][Medline]
  25. Visalli, R. J., J. Fairhurst, S. Kothandaraman, and A. Buklan. 2002. Characterization of the murine cytomegalovirus 38 kDa m137 gene product. Virus Res. 84:181-189.[CrossRef][Medline]
  26. Visalli, R. J., J. Fairhurst, S. Srinivas, W. Hu, B. Feld, M. DiGrandi, K. Curran, A. Ross, J. D. Bloom, M. van Zeijl, T. R. Jones, J. O'Connell, and J. I. Cohen. 2003. Identification of small-molecule compounds that selectively inhibit varicella-zoster virus replication. J. Virol. 77:2349-2358.[Abstract/Free Full Text]
  27. Wachsman, M., M. Kulka, C. C. Smith, and L. Aurelian. 2001. A growth and latency compromised herpes simplex virus type 2 mutant (ICP10DeltaPK) has prophylactic and therapeutic protective activity in guinea pigs. Vaccine 19:1879-1890.[CrossRef][Medline]
  28. Wang, K., L. Pesnicak, and S. E. Straus. 1997. Mutations in the 5' end of the herpes simplex virus type 2 latency-associated transcript (LAT) promoter affect LAT expression in vivo but not the rate of spontaneous reactivation of genital herpes. J. Virol. 71:7903-7910.[Abstract]
  29. Ward, P. L., and B. Roizman. 1994. Herpes simplex genes: the blueprint of a successful human pathogen. Trends Genet. 10:267-274.[CrossRef][Medline]
  30. Whitbeck, J. C., W. C. Lawrence, and L. J. Bello. 1994. Characterization of the bovine herpesvirus 1 homolog of the herpes simplex virus 1 UL24 open reading frame. Virology 200:263-270.[CrossRef][Medline]
  31. Yoshikawa, T., L. R. Stanberry, N. Bourne, and P. R. Krause. 1996. Downstream regulatory elements increase acute and latent herpes simplex virus type 2 latency-associated transcript expression but do not influence recurrence phenotype or establishment of latency. J. Virol. 70:1535-1541.[Abstract]

Journal of Virology, August 2005, p. 10498-10506, Vol. 79, No. 16
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.16.10498-10506.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:


This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Blakeney, S.
Right arrow Articles by Visalli, R. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Blakeney, S.
Right arrow Articles by Visalli, R. J.

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
J. Bacteriol. Mol. Cell. Biol. Microbiol. Mol. Biol. Rev.
Clin. Vaccine Immunol. ALL ASM JOURNALS