DNA immunization with a herpes simplex virus 2 bacterial artificial chromosome

Construction of a herpes simplex virus 2 (HSV-2) bacterial artificial chromosome (BAC) is described. BAC vector sequences were inserted into the thymidine kinase gene of HSV-2 by homologous recombination. DNA from cells infected with the resulting recombinant virus was transformed into E. coli, and colonies containing the HSV-2 BAC (HSV2-BAC) were isolated and analyzed for the expected genotype. HSV2-BAC DNA was infectious when transfected back into mammalian cells and the resulting virus was thymidine kinase negative. When used to immunize mice, the HSV2-BAC DNA elicited a strong HSV-2 specific antibody response that was equal to or greater than live virus immunization. Further, HSV2-BAC immunization was protective when animals were challenged with a lethal dose of virus. The utility of the HSV2-BAC for construction of recombinant virus genomes was demonstrated by elimination of the HSV-2 glycoprotein D (gD) gene. A recombinant HSV-2 BAC with the gD gene deleted was isolated and shown to be incapable of producing infectious virus following transfection unless an HSV gD gene was expressed in a complementing cell line. Immunization of mice with the HSV2 gD-BAC also elicited an HSV-2 specific antibody response and was protective. The results demonstrate the feasibility of DNA immunization with HSV-2 bacterial artificial chromosomes for replicating and nonreplicating candidate HSV-2 vaccines, as well as the utility of BAC technology for construction and maintenance of novel HSV-2 vaccines. The results further suggest that such technology will be a powerful tool for dissecting the immune response to HSV-2.     

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.     

A highly efficient protocol of generating and analyzing VZV ORF deletion mutants based on a newly developed luciferase VZV BAC system

Varicella Zoster Virus (VZV) is the causative agent for both varicella (chicken pox) and herpes zoster (shingles). As a member of the human herpesvirus family, VZV contains a large DNA genome, encoding 70 unique open reading frames (ORFs). The functions of the majority of these ORFs remain unknown. Recently, the full-length VZV (P-Oka strain) genome was cloned as a VZV bacteria artificial chromosome (BAC) and additionally a firefly luciferase cassette was inserted to generate a novel luciferase VZV BAC. In this study, a highly efficient protocol has been developed exploiting the new luciferase VZV BAC system to rapidly isolate and characterize VZV ORF deletion mutants by growth curve analysis in cell culture.     

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.     

Immunoselection of recombinant baculoviruses expressing high levels of biologically active herpes simplex virus type 1 glycoprotein D

Summary The DNA sequence encoding the complete herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) was inserted into a baculovirus transfer vector under control of the polyhedrin gene promoter of the baculovirusAutographa california nuclear polyhedrosis virus (AcNPV). After co-transfection ofSpodoptera frugiperda (Sf9) insect cells with wild-type AcNPV DNA and the recombinant transfer vector DNA, polyhedrin-negative recombinants that expressed high levels of HSV-1 gD were isolated using immunoaffinity selection with antibody coated magnetic particles followed by plaque purification. These recombinant baculoviruses expressed a protein that was slightly smaller than virion HSV-1 gD made in Vero cells. This recombinant protein was expressed at high levels. The expressed protein was glycosylated, was found on the membrane of Sf9 cells, and reacted with gD specific antibodies. Antibodies raised in mice to the recombinant gD neutralized HSV-1 as measured by plaque reduction assays. Mice inoculated with the recombinant baculovirus were completely protected from lethal challenge with HSV-1.     

Intertypic complementation and recombination between temperature-sensitive mutants of herpes simplex virus types 1 and 2

In an attempt to identify functionally “common” and “type-specific” genes in the genomes of herpes simplex virus types 1 (HSV-1) and 2 (HSV-2), intertypic complementation and recombination experiments between temperature-sensitive (ts) mutants of these viruses were undertaken. Intertypic complementation was carried out with seven HSV-1 ts mutants representing five complementation groups, and eight HSV-2 ts mutants representing five complementation groups. Efficient complementation was detected with most HSV-1 + HSV-2 ts mutant pairs, indicating extensive interchangeability of gene functions between HSV types 1 and 2. Progeny analysis of complementation yields demonstrated that complementation was symmetric; i.e., equal amounts of both HSV-1 and HSV-2ts mutants were produced. HSV-1 wild-type virus was also able to complement HSV-2 ts mutants efficiently. Intertypic recombination experiments demonstrated that heterologous mutant pairs which failed to complement also failed to recombine efficiently.     

HSV-1 and HSV-2 in herpes simplex encephalitis: A study of sixty-four cases in the United Kingdom

The incidence of herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2) in herpes simplex encephalitis (HSE) was investigated using cerebrospinal fluid (CSF) samples from sixty-four cases of HSE. A polymerase chain reaction (PCR) employing primers flanking a region of the HSV thymidine kinase gene common to both HSV-1 and HSV-2 was used to detect HSV in the CSF. HSV-1 and HSV-2 were differentiated by digestion with restriction enzymes. Two enzymes were employed; AvaI which cleaved only the HSV-2 gene product and AvaII which cleaved only the HSV-1 gene product. Sixty-three cases of HSE were found to be due to HSV-1; one case due to HSV-2. These data confirm previous observations that HSV-2 is a rare cause of post-neonatal herpes encephalitis but indicates that a PCR procedure capable of detection of both viruses is essential for efficient diagnosis of HSE. J. Med. Virol. 53:1–3, 1997. © 1997 Wiley-Liss, Inc.     

Herpes simplex virus 1 induced LOX-1 expression in an endothelial cell line, ECV 304

Infections, such as by Chlamydophilia pneumoniae, cytomegalovirus, herpes simplex virus, and Helicobacter pylori, have been shown to be involved in atherogenesis. Herpes simplex virus I (HSV-1) could infect vascular endothelial cells, and it has been shown that, when endothelial cells were activated with oxidized LDL (oxLDL), a number of cellular events are occurred, leading to endothelial cell dysfunction. Since LOX-1 is a major receptor for oxLDL on endothelial cells and its expression was increased in atherosclerosis, we investigated whether HSV1 infection can lead to the increase expression of LOX-1 in endothelial cells. LOX-1 mRNA expression determined by RT-PCR and LOX-1 promoter activity measured by luciferase assay were increased in endothelial cells following HSV-1 infection. This suggests that one of the mechanisms by which HSV-1 is involved in atherogenesis maybe the enhanced uptake of oxLDL via the increased expression of LOX-1 in endothelial cells.     

Expression of human immunodeficiency virus type 1gag gene using genetically engineered herpes simplex virus type 1 recombinants

Infectious herpes simplex virus type 1 (HSV-1) recombinants were constructed by inserting the cDNA sequence of the human immunodeficiency virus type 1 (HIV-1)gag gene (from nucleotide position 675 [SacI] to 3859 [Asp 718] of the cDNA sequences of HIV-1 strain BH-10) within the DNA sequences of theBamHI DNA fragment B of the genome of an apathogenic HSV-1 strain HFEM. This HSV-1 strain possesses a 4.1-kbp deletion within theBamHI DNA fragment B between 0.762 and 0.789 map units of the viral genome, which allows the insertion of at least 4 kbp of foreign genetic material into this particular region. The DNA sequences of the immediate early promoter (IE4) of HSV-1 that were inserted upstream from thegag gene were used as a promoter. The screening of 205 virus stocks derived from individual plaques revealed that 46 recombinant viruses harbor HIV-1gag-specific DNA sequences. However, it was found that only six of the recombinant viruses are able to express thegag gene product of HIV-1. This indicates that the ratio of the positive recombination events is about 2.9%.     

Topical application of polyethylenimine as a candidate for novel prophylactic therapeutics against genital herpes caused by herpes simplex virus

Herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) cause genital herpes, which can enhance the acquisition of human immunodeficiency virus. The development of anti-HSV agents with novel mechanisms of action is urgently required in the topical therapy of genital herpes. In this study, the in vitro and in vivo anti-HSV effects of Epomin SP-012^®, a highly cationic polyethylenimine, were evaluated. When the in vitro antiviral effects of SP-012 were assessed, this compound showed potent activity against HSV-1 and HSV-2. It inhibited the attachment of HSV-2 to host cells and cell-to-cell spread of infection in a concentration-dependent manner and exerted a virucidal effect. No SP-012-resistant HSV-2 was found when the virus was successively passaged in the presence of SP-012. In a mouse genital herpes model, topically administered SP-012 inhibited the progression of the disease caused by HSV infection. These data illustrate that SP-012 may be a novel class of HSV inhibitor that would be acceptable for long-term topical application.     

Further characterization of virus obtained from herpes simplex virus type 1 recurrences and primary infections. Influence of the temperature of incubation upon glycoprotein synthesis and virus release

Summary The virus contained in clinical isolates of herpes simplex virus type 1 (HSV-1) which have not undergone previousin vitro passages (new isolates) differs from HSV-1 prototype strains with respect to infected cell glycoprotein pattern, and, most probably efficiency of virus egress at 37° C. The differences can be abolished by lowering the temperature of incubation to 33° C. A few tissue culture passages cause the conversion of the original virus to a virus undistinguishable from HSV-1 prototype strains with respect to the parameters mentioned above.With 2 Figures     

Importance of theHpaI-P sequence for herpes simplex virus-1 replication in the adrenal glands

Summary The ability of several strains and recombinants of herpes simplex virus 1 (HSV-1) to proliferate in the adrenal glands and to invade the spinal cord was studied. After intraperitoneal infection, pathogenic HSV-1 strains replicated in the adrenal glands, penetrated the spinal cord and migrated to the brain. The nonpathogenic strain HFEM could not replicate in the adrenal glands, but the recombinant virus MLC1 was able to do so after rescue by reinsertion of theHpaI-P sequence into theBamHI fragment of HFEM DNA. However the recombinant MLC1 virus could not penetrate the spinal cord.The effect of HSV-1 infection on the expression of the cellular genes for multidrug resistance (in the adrenal glands) and proenkephalin A (in the spinal cord) was also studied.     

Herpes Simplex Virus 1 Suppresses the Function of Lung Dendritic Cells via Caveolin-1

Caveolin-1 (Cav-1), the principal structural protein of caveolae, has been implicated as a regulator of virus-host interactions. Several viruses exploit caveolae to facilitate viral infections. However, the roles of Cav-1 in herpes simplex virus 1 (HSV-1) infection have not fully been elucidated. Here, we report that Cav-1 downregulates the expression of inducible nitric oxide synthase (iNOS) and the production of nitric oxide (NO) in dendritic cells (DCs) during HSV-1 infection. As a result, Cav-1 deficiency led to an accelerated elimination of virus and less lung pathological change following HSV-1 infection. This protection was dependent on iNOS and NO production in DCs. Adoptive transfer of DCs with Cav-1 knockdown was sufficient to confer the protection to wild-type (WT) mice. In addition, Cav-1 knockout (KO) (Cav-1^−/−) mice treated with an iNOS inhibitor exhibited significantly reduced survival compared to that of the nontreated controls. We found that Cav-1 colocalized with iNOS and HSV-1 in caveolae in HSV-1-infected DCs, suggesting their interaction. Taken together, our results identified Cav-1 as a novel regulator utilized by HSV-1 to evade the host antiviral response mediated by NO production. Therefore, Cav-1 might be a valuable target for therapeutic approaches against herpesvirus infections.     

Genomic characterization of two predominant genotypes of herpes simplex virus type 1

Summary Genomic profiles of 66 strains of herpes simplex virus type 1 (HSV-1) isolated in Japan were investigated with regard to restriction fragment length polymorphism (RFLP) and length variation of fragments containing reiterations. There were two predominant genotypes of F1 and F35, and the genomic characteristics of each were studied. The nucleotide change between F1 and F35 was estimated to be 1.5%. An RFLP marker (VR23) peculiar to genotype F35 was identified as the first case of genomic marker specific to a predominant genotype of HSV-1, and is the diagnostic marker of F35. Thea sequences (repeating in an HSV-1 genome and containing reiterations) of F35 were cleaved by Sac II on the DR4 (direct repeat 4) stretch, whilea sequences of F1 had a rearranged DR4 and were resistant to Sac II digestion. Thus, analyses of fragments containing reiterations, such asa sequences, can serve to classify HSV-1 strains as well as for purpose of differentiation. The proportion of strains derived from primary infection to those from recurrent infection was higher in strains of F35 than in those of F1, and this genotypic difference within HSV-1 may possibly influence clinical manifestations.     

Computer-assisted primary and secondary structure analyses of DNA polymerases of herpes simplex,Epstein-Barr and varicella zoster viruses reveal conserved domains with some homology to DNA-binding domain in E. coli DNA pol I

The primary and secondary structure of herpes simplex virus type 1 (HSV-1), varicella-zoster (VZV) and Epstein-Barr virus (EBV) DNA polymerases was calculated by means of computer programs. The comparison of HSV-1 polymerase (pol) sequence to the known primary and tertiary structure of E. coli DNA pol I revealed five short homologous sequences, one of which coincided with the -helical structure of the DNA-binding domain of E. coli DNA pol. Comparison by primary and secondary structure computer programs of the three DNA polymerases coded by herpesviruses HSV-1, VZV and EBV led to the identification of polypeptide sequences shared by the three DNA pols. In a similar way, the secondary structure of the DNA pol polypeptide in the vicinity of the mutation leading to PAA resistance in HSV-1 DNA pol helped to identify the role of this sequence in the binding of phosphate donated by the nucleoside triphosphate molecule which binds to the DNA pol. Although the computer secondary structure programs are about 60% accurate, it was possible to obtain new information on the properties of certain domains in the DNA polymerases of HSV-1, VZV and EBV.Abbreviations aa amino acids - EBV Epstein-Barr virus - HSV-1 herpes simplex virus type 1 - M_r molecular weight - PAA phosphonoacetic acid - PFA phosphonoformic acid - pol polymerase - VZV varicella-zoster virus Requests for reprints should be addressed to Professor Yechiel Becker, Department of Molecular Virology, Faculty of Medicine, The Hebrew University, P.O. Box 1172, 91010 Jerusalem, Israel.     

The association between serum 25-hydroxyvitamin D and the prevalence of herpes simplex virus

Previous studies have reported a potential anti-infection effect for vitamin D. However, the relationship between vitamin D status and herpes simplex virus (HSV) infection has not yet been evaluated. Therefore, this study aimed to determine the association between serum 25-hydroxyvitamin D [25(OH)D] and infection with HSV types 1 and 2 (HSV-1 and HSV-2). Data were collected from the National Health and Nutrition Examination Survey from 2007 to 2016. The association between 25(OH)D and HSV prevalence was evaluated using propensity score matching (PSM) and univariate and multivariate logistic regression analyses. Overall, 14 174 participants were included in the final analysis. Before PSM, 8639 (60.9%) had positive HSV-1 and 2636 (18.6%) had HSV-2. The HSV-1 and HSV-2 positive groups had more females and older individuals (p < 0.05). The HSV-2 patients had lower 25(OH)D levels than those with HSV-1. Age and gender did not differ in the groups after PSM (p > 0.05). The 25(OH)D level was significantly lower in the HSV-1 and HSV-2 groups than in the non-HSV infection groups. Multivariate logistic regression showed that serum 25(OH)D level was negatively associated with HSV-1 and HSV-2 infection (odds ratio [OR] = 0.730 and 0.691, p < 0.001, respectively). Vitamin D deficiency was an independent risk factor for both HSV-1 and HSV-2 (adjusted OR = 2.205 and 2.704, p < 0.001, respectively). Lower serum 25(OH)D levels correlated significantly with increased HSV-1 and HSV-2 infection risk.     

Enhancement of herpes simplex virus type 2 (HSV-2) DNA synthesis in infected cells that constitutively express the BglII-N region of the HSV-2 genome

The BglII-N fragment of the herpes simplex virus type-2 (HSV-2) genome encodes one of two known transforming regions of this DNA virus. In this study, we report the derivation of HeLa S3 cells (2DC₄) that stably express the HSV-2 BglII-N region, including the small subunit of HSV-2 ribonucleotide reductase (RR). Superinfection of the 2DC₄ cells with wild-type HSV-2 resulted in the efficient induction of HSV-2-encoded ICP10, DNA polymerase, and thymidine kinase. The amount of HSV-2 DNA synthesis in 8-hr HSV-2-infected 2DC₄ cells was enhanced 2.6±0.6-fold relative to infected control cells. Furthermore, the replication kinetics of HSV-2 DNA in 2DC₄ cells were accelerated relative to HeLa S3 cells; HSV-2 DNA synthesis was detectable as early as 3 hr postinfection in 2DC₄ cells as compared to 6 hr postinfection in HeLa S3 cells. These results suggest that the BglII-N region of HSV-2 encodes function(s) that activate the viral DNA synthesis apparatus and that this activation could relate to the transforming ability of this DNA region.     

Sensitive and rapid detection of herpes simplex virus and varicella-zoster virus DNA by loop-mediated isothermal amplification

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method in which reagents react rapidly and efficiently, with a high specificity, under isothermal conditions. We used a LAMP assay for the detection of herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), and varicella-zoster virus (VZV). The virus specificities of primers were confirmed by using 50 HSV-1, 50 HSV-2, and 8 VZV strains. The assay was performed for 45 min at 65 degrees C. The LAMP assay had a 10-fold higher sensitivity than a PCR assay. An analysis of nucleotide sequence variations in the target and primer regions used for the LAMP assay indicated that 3 of 50 HSV-1 strains had single nucleotide polymorphisms. No HSV-2 or VZV strains had nucleotide polymorphisms. Regardless of the sequence variation, there were no differences in sensitivity with the HSV-1-specific LAMP assay. To evaluate the application of the LAMP assay for clinical diagnosis, we tested clinical samples from 40 genital herpes patients and 20 ocular herpes patients. With the LAMP assay, 41 samples with DNA extraction and 26 direct samples without DNA extraction were identified as positive for HSV-1 or HSV-2, although 37 samples with DNA extraction and just one without DNA extraction were positive by a PCR assay. Thus, the LAMP assay was less influenced than the PCR assay by the presence of inhibitory substances in clinical samples. These observations indicate that the LAMP assay is very useful for the diagnosis of HSV-1, HSV-2, and VZV infections.     

Suitability and clinical application of a multiplex nested PCR assay for the diagnosis of herpes simplex virus infections

A novel multiplex nested polymerase chain reaction (PCR) assay was designed and evaluated for routine diagnosis of herpes simplex virus (HSV) infections in patients with either putative HSV infection of the central nervous system or suspected HSV keratitis. Single-tube amplification of HSV type 1 (HSV-1) or type 2 (HSV-2) DNA extracted from cerebrospinal fluid (CSF) or from keratectomy specimens was followed by differentiation of the virus type-specific PCR products either by agarose gel analysis or by DNA enzyme immunoassay. Among 417 CSF specimens obtained from 395 consecutive patients with clinically suspected HSV infection, 11 (2.6%) were positive for HSV-1 DNA and four (1.0%) probes were positive for HSV-2 DNA. None of the specimens was positive for both HSV-1 and HSV-2 DNA. The genome of HSV-2 was detected in a CSF sample obtained from a woman with meningoencephalitis and genital herpes. The presence of PCR inhibitors was detected in six of 111 (5.4%) reconstructed CSF samples. Inhibition could be removed following extraction with a commercial kit. HSV-1 DNA, but no HSV-2 DNA, was detected in corneal buttons obtained from patients with suspected herpetic keratitis. No contamination has been recorded during the 2-year routine use of this test, which has met the specific requirements of a diagnostic laboratory. © 1996 Wiley-Liss, Inc.