Human Simplex Virus Research Articles

Preparation of BFV Gag antiserum and preliminary study on cellular distribution of BFV

Viruses (e.g. Human immunodeficiency virus, Human simplex virus and Prototype foamy virus) are obligate intracellular parasites and therefore depend on the cellular machinery for cellular trafficking. Bovine foamy virus (BFV) is a member of the Spumaretrovirinae subfamily of Retroviruses, however, details of its cellular trafficking remain unknown. In this study, we cloned the BFV gag gene into prokaryotic expression vector pET28a and purified the denaturalized Gag protein. The protein was used to immunize BALB/c mouse to produce antiserum, which could specifically recognize the BFV Gag protein in BFV-infected cells through western blot assay. Additionally, these results demonstrated that both the optimal and suboptimal cleavage of Gag protein occur in BFV-infected cells. Subsequently, the Gag antiserum was used to investigate subcellular localization of BFV. In immunofluorescence microscopy assays, colocalization microtubules (MTs) and assembling viral particles were clearly observed, which implied that BFV may transport along cellular MTs in host cells. Furthermore, MTs-depolymerizing assay indicated MTs were required for the efficient replication of BFV. In conclusion, our study suggests that BFV has evolved the mechanism to hijack the cellular cytoskeleton for its replication.

Association of varicella zoster virus load in the aqueous humor with clinical manifestations of anterior uveitis in herpes zoster ophthalmicus and zoster sine herpete

Aim:To investigative whether clinical manifestations of anterior uveitis are associated with the viral load of varicella zoster virus (VZV) in the aqueous humor in patients with herpes zoster ophthalmicus (HZO)...

Submassive hepatocellular necrosis associated with infiltration by peripheral T‐cell lymphoma of cytotoxic phenotype: Report of two cases

Hepatocellular necrosis may be complicated in the clinical course of T-cell lymphoma. Herein are reported two cases of peripheral T-cell lymphoma associated with a rapid increase of serum transaminases in their final course. The final diagnoses were enteropathy-type T-cell lymphoma of the jejunum (patient 1) and hepatosplenic T-cell lymphoma (patient 2). The livers of both patients had non-zonal, well-demarcated coagulation necrosis (approximately 70% in patient 1 and 40% in patient 2) that was infiltrated by lymphoma cells bearing a cytolytic granule content (granzyme B). Hepatocytes in the necrotic areas in patient 2 showed immunoreactivity for human simplex virus type 1. Although the mechanism of the hepatocyte necrosis is unclear, it is suggested that the hepatocyte submassive necrosis may be related to an infection complicated in the final course of lymphoma or by certain direct effects of the tumor cells.

A Comparative Study of Uracil-DNA Glycosylases from Human and Herpes Simplex Virus Type 1

Uracil-DNA glycosylase (UNG) is the key enzyme responsible for initiation of base excision repair. We have used both kinetic and binding assays for comparative analysis of UNG enzymes from humans and herpes simplex virus type 1 (HSV-1). Steady-state fluorescence assays showed that hUNG has a much higher specificity constant (k(cat)/K(m)) compared with the viral enzyme due to a lower K(m). The binding of UNG to DNA was also studied using a catalytically inactive mutant of UNG and non-cleavable substrate analogs (2'-deoxypseudouridine and 2'-alpha-fluoro-2'-deoxyuridine). Equilibrium DNA binding revealed that both human and HSV-1 UNG enzymes bind to abasic DNA and both substrate analogs more weakly than to uracil-containing DNA. Structure determination of HSV-1 D88N/H210N UNG in complex with uracil revealed detailed information on substrate binding. Together, these results suggest that a significant proportion of the binding energy is provided by specific interactions with the target uracil. The kinetic parameters for human UNG indicate that it is likely to have activity against both U.A and U.G mismatches in vivo. Weak binding to abasic DNA also suggests that UNG activity is unlikely to be coupled to the subsequent common steps of base excision repair.

The effect of cell killing and apoptosis by human herpes simplex virus- thymidine kinase/ganciclovir system combined with allitride in BIU87 cells

To study the killing effect of human herpes simplex virus-thymidine kinase/ganciclovir (HSV-TK/GCV) system combined with allitride and the possible apoptosis mechanism in BIU87 cells. The cytotoxicity after combination were estimated by theamine blue tetrazolium bromide (MTT). The morphological changes were observed with inverted microscope and in-situ cell apoptosis detection kit. Changes of apoptosis rate and cell cycle were assessed by flow cytometry. B-cell lymphoma-2 (bcl-2), bax, caspase-3 (cysteine aspartate specific proteinase) mRNA changes were detected by reverse transcriptase polymerase chain reaction, and caspase-3 activity was estimated with colorimetry. For combination group, the cell killing rate was raised to 72.50% to compare with 35.00% of GCV and 37.00% of allitride separately and there was a synergistic effect between these two drugs. The cell apoptosis was induced in all three groups and for the combination group the time of S-phase and G(2)-phase arrest were earlier than other two groups. Both drugs could inhibit the expression of bcl-2 and promote the expression and activity of caspase-3. The combination of HSV-TK/GCV system with allitride can inhibit the proliferation of BIU87 cells congenerously through apoptosis, which may be correlated with S- and G(2)-phase arrest, down-regulation of bcl-2 and increased caspase-3 expression and its activity.

Complete genome sequence of cercopithecine herpesvirus 2 (SA8) and comparison with other simplexviruses

We have obtained the complete sequence of the herpesvirus simian agent 8 (SA8; cercopithecine herpesvirus 2) a baboon simplexvirus closely related to the monkey B virus and herpes simplex virus types 1 and 2. The genome of SA8 is 150,715 bp long, with an overall G/C content of 76%, the highest among the simplexviruses sequenced so far. The sequencing has confirmed that the genomic arrangement of SA8 is similar to that of other simplexviruses: unique long and unique short regions bordered by two sets of inverted repeats. All genes identified in SA8 are homologous and collinear with those of the monkey B virus, including the lack of the RL1 open reading frame, a gene responsible for neurovirulence in human herpes simplex viruses. This latter finding supports the hypothesis that a different pathogenetic mechanism may have developed in human simplexviruses, after their divergence from monkey simplexviruses.

Characterization and expression of the pseudorabies virus early gene UL54

Pseudorabies virus (PRV) is an alphaherpesvirus, and its gene organization and regulation are similar to the well-characterized human simplex virus (HSV). Sequence analysis of the complete coding region of PRV UL54 gene revealed that the UL54 gene consisted of 1092 nucleotides encoding a protein of 363 amino acids and the gene showed homology to HSV immediate-early protein ICP27. Detection of the UL54 transcript in infected cells by reverse transcription-polymerase chain reaction (RT-PCR) demonstrated that the UL54 gene belonged to the early kinetic class based on sensitivity to cycloheximide and insensitivity to phosphonoacetic acid (PAA). To study the structure and function of UL54 protein, this gene was subcloned on Escherichia coli expression vector pET28b for overexpression, and the expressed product was applied to generate specific antibody against UL54 protein. The specificity of the mouse immuneserum was confirmed by its ability to react with a 40 kDa viral protein present in the PRV infected cells in Western immunblotting assay, detected as early as 4 h after infection. In addition, immunoperoxidasing staining of PRV infected cells undertaken with this antibody demonstrated mainly nuclear staining pattern. Furthermore, the RNA binding potential of UL54 protein was demonstrated by its binding activity to poly(G) RNA homopolymer in Northwestern blotting assay.

Characterization and expression of the pseudorabies virus early gene UL54

Pseudorabies virus (PRV) is an alphaherpesvirus, and its gene organization and regulation are similar to the well-characterized human simplex virus (HSV). Sequence analysis of the complete coding region of PRV UL54 gene revealed that the UL54 gene consisted of 1092 nucleotides encoding a protein of 363 amino acids and the gene showed homology to HSV immediate-early protein ICP27. Detection of the UL54 transcript in infected cells by reverse transcription-polymerase chain reaction (RT-PCR) demonstrated that the UL54 gene belonged to the early kinetic class based on sensitivity to cycloheximide and insensitivity to phosphonoacetic acid (PAA). To study the structure and function of UL54 protein, this gene was subcloned on Escherichia coli expression vector pET28b for overexpression, and the expressed product was applied to generate specific antibody against UL54 protein. The specificity of the mouse immuneserum was confirmed by its ability to react with a 40 kDa viral protein present in the PRV infected cells in Western immunblotting assay, detected as early as 4 h after infection. In addition, immunoperoxidasing staining of PRV infected cells undertaken with this antibody demonstrated mainly nuclear staining pattern. Furthermore, the RNA binding potential of UL54 protein was demonstrated by its binding activity to poly(G) RNA homopolymer in Northwestern blotting assay.

In vivo imaging of HIV protease activity in amplicon vector-transduced gliomas.

In vivo imaging of endogenously expressed mammalian proteases has been useful for the detection of cancer and preneoplastic lesions, for staging of inflammatory and autoimmune diseases, and for testing the efficacy of novel protease inhibitors. Here we report on the synthesis of a novel imaging probe that is specific for HIV-1 protease (PR). The probe was designed to be biocompatible, i.v. injectable, and detectable by fluorescence imaging. Human Gli36 glioblastoma cells infected with an human simplex virus amplicon vector expressing HIV-1PR showed specific fluorescence activation, an effect that could be inhibited by the HIV-1PR inhibitor, indinavir. The transfer of the HIV-1PR marker gene could be detected in vivo after intratumoral delivery of the human simplex virus-amplicon vector. These results are the first proof of principle that viral proteases can directly be imaged in vivo. These findings may be directly applicable in using viral protease expression as a transgene marker in tumor therapy and may have implications in testing the efficacy of HIV-1PR inhibitors in vivo.

Chlamydia trachomatis, herpes simplex virus 2, and human T-cell lymphotrophic virus type 1 are not associated with grade of cervical neoplasia in Jamaican colposcopy patients.

A few recent studies have suggested that other sexually transmitted infections may increase the likelihood of a human papillomavirus (HPV) infection progressing to high-grade cervical neoplasia and cancer. The goal was to assess whether exposures to Chlamydia trachomatis, human T-cell lymphotrophic virus type 1 (HTLV-I), and/or human simplex virus type 2 (HSV-2) are greater in colposcopy patients with cervical intraepithelial neoplasia grade 3 or cancer (CIN3+) than in patients with low-grade cervical neoplasia (CIN1). Sequential patients (n=447) attending a colposcopy clinic in Kingston, Jamaica, a country with high cervical cancer rates and high HTLV-I prevalence, were tested for (1) HPV DNA by L1 consensus primer (MY09/11) polymerase chain reaction assays, (2) C trachomatis DNA by ligase chain reaction, (3) C trachomatis antibodies by both microimmunofluorescence and a peptide (VS4) enzyme linked immunosorbent assay (ELISA), (4) HTLV-I antibodies by ELISA confirmed by western blotting, and (5) HSV-2 antibodies by a recombinant HSV-2-specific ELISA. Odds ratios and 95% confidence intervals were estimated with use of multinomial logistic regression models. HPV DNA detection was associated with grade of cervical neoplasia but other evaluated sexually transmitted infections were not. HTLV-I, C trachomatis, and/or HSV-2 were not associated with severity of cervical neoplasia in Jamaican women.

The contrasting IgG-binding interactions of human and herpes simplex virus Fc receptors.

A virally encoded, high-affinity Fc receptor (FcR) is found on herpes simplex virus type 1 (HSV-1) particles and infected cells where its binding of non-immune IgG protects cells from host-mediated lysis. Whilst mutation or aglycosylation of the IgG CH2 domain reduced binding to human FcR, the interaction with HSV-1 FcR was not affected. However, the HSV-1 FcR, unlike human FcR, discriminates between human IgG1 allotypes, being sensitive to changes at positions 214 (CH1) and 356/358 (CH3), away from its proposed binding site at the CH2-CH3 interface. The biological consequences are not known but this is the first evidence of a major functional difference between IgG1 allotypes.

The contrasting IgG-binding interactions of human and herpes simplex virus Fc receptors

The contrasting IgG-binding interactions of human and herpes simplex virus Fc receptors

5-(Trifluoromethyl)-β- l-2′-deoxyuridine, the l-Enantiomer of Trifluorothymidine: Stereospecific Synthesis and Antiherpetic Evaluations

As a part of our ongoing work on β- l-nucleoside analogues as potential antiviral drugs, we have synthesized 5-(trifluoromethyl)-β- l-2′-deoxyuridine ( l-TFT), the hitherto unknown l-enantiomer of trifluorothymidine (CF 3dUrd, TFT). We have also studied the effect of l-TFT on human and herpes simplex virus (HSV) type 1 and 2 thymidine kinases, and human thymidine phosphorylase, as well as its anti-HSV-1 and anti-HSV-2 activities in cell cultures. l-TFT has been found: (i) to inhibit HSV-1 TK with activity comparable to TFT, with no effect on human TK, (ii) to be phosphorylated by the viral enzyme with similar efficiency to TFT, (iii) to be resistant, in contrast to TFT, to hydrolysis by human thymidine phosphorylase. Unfortunately, when evaluated in cell cultures, l-TFT did not show any anti-HSV-1 and anti-HSV-2 activities.

Prevalence of high-grade dysplasia and cancer in the anal canal in human papillomavirus-infected individuals.

The incidence of anal cancer is higher in patients with anal canal condyloma, a sexually transmitted disease, than in the general population. We determined the prevalence of anal dysplasia and cancer in patients with anal canal condyloma with respect to human immunodeficiency virus (HIV) status, immunity status, and human papillomavirus types. In 174 consecutive patients (114 HIV positive, 60 HIV negative) with anal canal condyloma, lesions were cured, and the patients were then followed up prospectively. Langerhans cells (LCs) in normal anal mucosa were quantified, and viruses (Epstein-Barr virus, cytomegalovirus, human simplex virus 1, and various human papillomavirus [HPV] types) were characterized on inclusion. During follow-up (median 26 months), relapsed condylomas were resected and examined histologically. HIV load and CD4 T-lymphocyte counts in serum were determined at each visit. Several factors differed significantly between HIV-positive and HIV-negative patients: LCs/mm anal tissue (15 vs. 30), oncogenic HPV (27% vs. 13%), other current anal infections (44% vs. 0%), and sex ratio (93% vs. 73% male). During follow-up, condylomas relapsed in 75% of the HIV-positive patients, with 19 high-grade dysplasias (HGDs) and 1 invasive carcinoma, but in only 6% of HIV-negative patients, with 1 HGD. Male sex, HIV positivity, and <15 LCs/mm tissue were independent risk factors for condyloma relapse. HIV positivity, HGD before inclusion, and condyloma relapse were independent risk factors for HGD and cancer. Serum HIV load was associated with relapse, whereas CD4 T-lymphocyte counts were not. The prevalence of HGD and carcinoma is higher in HIV-positive than in HIV-negative patients, probably because of HPV activity. HIV-positive patients with high serum HIV load and/or a history of anal dysplasia should be examined by anoscopy, and condylomas should be analyzed histologically.

Cloning and Characterization of the Multisubstrate Deoxyribonucleoside Kinase of Drosophila melanogaster

A Drosophila melanogaster deoxyribonucleoside kinase (Dm-dNK) was reported to phosphorylate all four natural deoxyribonucleosides as well as several nucleoside analogs (Munch-Petersen, B., Piskur, J., and Sondergaard, L. (1998) J. Biol. Chem. 273, 3926-3931). The broad substrate specificity of this enzyme together with a high catalytic rate makes it unique among the nucleoside kinases. We have in the present study cloned the Dm-dNK cDNA, expressed the 29-kDa protein in Escherichia coli, and characterized the recombinant enzyme for the phosphorylation of nucleosides and clinically important nucleoside analogs. The recombinant enzyme preferentially phosphorylated the pyrimidine nucleosides dThd, dCyd, and dUrd, but phosphorylation of the purine nucleosides dAdo and dGuo was also efficiently catalyzed. Dm-dNK is closely related to human and herpes simplex virus deoxyribonucleoside kinases. The highest level of sequence similarity was noted with human mitochondrial thymidine kinase 2, and these enzymes also share many substrates. The cDNA cloning and characterization of Dm-dNK will be the basis for studies on the use of this multisubstrate nucleoside kinase as a suicide gene in combined gene/chemotherapy of cancer.

Topically Applied Human Recombinant Monoclonal IgG1Antibody and Its Fab and F(ab′)2Fragments Protect Mice from Vaginal Transmission of HSV-2

A recombinant human anti-herpes simplex virus monoclonal IgG1antibody and the corresponding Fab and F(ab′)2fragments were tested for efficacy in preventing vaginal transmission of HSV-2 infection in a well-established mouse model for genital herpes. IgG1, Fab, and F(ab′)2were approximately equally protective; vaginal delivery of 1–5 ng provided approximately 50% protection, and vaginal delivery of 400 ng completely protected mice from genital herpes infection (P< 0.001). These results suggest that topical applications of human monoclonal antibodies may be useful in developing new methods for preventing sexually transmitted disease.

Infection concomitant with pediatric renal allograft rejection.

Renal allograft rejection episodes are frequent in children and often lead to allograft failure. Frequent association of fever with rejection in our transplant program provoked a prospective evaluation of concurrent infection during rejection episodes. Because cytomegalovirus has an established role in rejection and allograft survival, evaluation of cytomegalovirus and other herpes viruses (human simplex virus type 1, varicella, Epstein-Barr virus, and human herpes virus type 6 [HHV-6]) was undertaken in addition to standard bacterial investigation. A total of 37 patients were followed over a 30-month period. Six of eight rejection episodes were associated with herpes viruses (HHV-6, n = 6, and Epstein-Barr virus, n = 1). Three of the herpes-group-associated rejection episodes were treated with antiviral therapy in addition to pulse steroid treatment, with full recovery. The three patients with HHV-6-associated rejection episodes who were treated with pulse steroids, but no antiviral therapy, developed chronic allograft rejection. The recipient's response to allograft antigens may be influenced by concomitant herpes infection, and specific antiviral therapy appears to be indicated when infection is confirmed in association with rejection. An antiviral treatment program coupled with modulation of standard antirejection immunotherapy has the potential to improve morbidity and mortality in the pediatric renal transplant population.

Recurring episodes of meningitis (Mollaret's meningitis) with one showing an association with herpes simplex virus type 2

Benign recurrent aseptic (Mollaret's) meningitis is a rare disease of unknown aetiology. We report the case of a 27-year-old woman who experienced three episodes of lymphocytic meningitis. Human Simplex Virus (HSV) type 2 DNA was detected in the CSF by PCR amplification indicating the diagnosis of recurrent HSV type 2 meningitis. Our observation suggests that search of herpes virus DNA by PCR amplification on CSF may be useful in unexplained recurrent meningitis.

Herpes simplex virus-infected human fibroblasts are resistant to and inhibit cytotoxic T-lymphocyte activity.

We examined the ability of human anti-herpes simplex virus (HSV) cytotoxic T lymphocytes (CTL) to lyse autologous human fibroblasts infected with HSV. In contrast to HSV-infected human Epstein-Barr virus-transformed B cells (LCL), which were lysed by HLA-restricted anti-HSV CTL, autologous fibroblasts infected with HSV were resistant to lysis. This resistance was not due to a lack of infectivity or production of HSV proteins since greater than 90% of the cells were infected and expressed abundant levels of viral proteins. HSV-infected human fibroblasts were also tested for susceptibility to lysis by alloantigen-specific CTL. Although allogeneic LCL and uninfected allogeneic fibroblasts were killed, human fibroblasts infected with HSV demonstrated a time-dependent resistance to lysis by alloantigen-specific CTL. HSV-infected human fibroblasts were not resistant to all forms of cell-mediated cytotoxicity since they were sensitive to antibody-dependent cellular cytotoxicity. Although one may suspect that the resistance of HSV-infected human fibroblasts to anti-HSV CTL and alloantigen-specific CTL-mediated lysis was due to a lack of major histocompatibility complex expression, Confer et al. (Proc. Natl. Acad. Sci. USA 87:3609-3613, 1990) previously demonstrated that incubation of human natural killer and lymphokine-activated killer cells with monolayers of human fibroblasts infected with HSV "disarmed" the killers in that they were unable to lyse sensitive target cells. We extend their results and show that incubation of anti-HSV CTL or alloantigen-specific CTL with uninfected fibroblasts did not affect their lytic activity, whereas CTL incubated with HSV-infected fibroblasts for 2 to 6 h rendered the CTL incapable of lysing their normally sensitive target cells. Indeed, human fibroblasts infected for merely 2 h with HSV were able to profoundly inhibit the cytotoxic activity of alloantigen-specific CTL. Thus, HSV-infected human fibroblasts are not inherently resistant to lysis by anti-HSV CTL or alloantigen-specific CTL, but rather contact of CTL with HSV-infected fibroblasts resulted in inactivation of the CTL. The inactivation of CTL appears to be HSV specific since incubation of alloantigen-specific CTL in sandwich assays with fibroblasts infected with HSV type 1 (HSV-1) or HSV-2 resulted in inactivation, whereas incubation of CTL with fibroblasts infected with adenovirus or vaccinia virus had no effect. Further, although incubation of alloantigen-specific CTL in sandwich assays with HSV-infected fibroblasts resulted in inhibition of CTL activity, exposure of CTL in Transwell cultures to cell-free supernatant from HSV-infected fibroblasts did not mediate this inhibitory effect.(ABSTRACT TRUNCATED AT 400 WORDS)

Simian alphaherpesviruses and their relation to the human herpes simplex viruses

Biochemical and immunological properties of structural and non-structural polypeptides of the human simplex viruses (HSV1 and HSV2) and four related herpesviruses of non-human primates [Herpesvirus simiae (B virus), H. cercopithicus (SA8), H. saimiri 1 (HVS 1), and H. ateles 1 (HVA 1)] were compared. Using a radioimmunoassay (RIA), the presence of antigenic determinants shared among all six viruses was demonstrated. The relative degree of antigenic cross-reactivity among these viruses was further assessed by competition RIA. Antigenically, HSV 1 and HSV 2 were most closely related to each other although both SA 8 and B virus were also very closely related to HSV 1. Considerably less cross-reactivity existed between either HVS 1 or HVA 1 and the other four primate herpesviruses. Cross-hybridization between simian and human herpesvirus genomes demonstrated that extensive homology exists between each of the simian viruses and both HSV1 and HSV 2. Viral polypeptides bearing common antigenic determinants were identified by immune precipitation of infected cell polypeptides and by immunoblotting. Among the polypeptides of HSV which were recognized by antisera to simian viruses were the VP 5 and p40 proteins, both of which are structural components of the virion nucleocapsid. Using recombinant plasmids containing sequences of the HSV 1 VP5, p40, DNA polymerase, major DNA binding protein, and TK enzyme genes, homologous sequences were detected in all four simian viruses. Together, these results demonstrate that HSV 1, HSV 2, SA 8, and B virus form a closely related sub-group of the primate herpesviruses; HVS 1 and HVA 1 are also related to the other four primate herpesviruses, albeit more distantly.