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.     

Evaluation of mixed infection cases with both herpes simplex virus types 1 and 2

Herpes simplex virus type 1 (HSV-1) is isolated principally from the upper half of the body innervated by the trigeminal ganglia whereas herpes simplex virus type 2 (HSV-2) is generally isolated from the lower half of the body innervated by the sacral ganglia. However, recent reports suggest that HSV-1 and HSV-2 can each infect both the upper and lower half of the body causing a variety of symptoms and there is a possibility that HSV-1 and HSV-2 infections can occur simultaneously with both causing symptoms. HSV type in clinical isolates from 87 patients with genital herpes and 57 with ocular herpes was determined by the polymerase chain reaction (PCR), and six cases of mixed infection with both HSV-1 and HSV-2 were identified. Of the six cases, three were patients with genital herpes and three were ocular herpes patients. Analysis of the copy number of the HSV-1 and HSV-2 genome by a quantitative real time PCR demonstrated that HSV-1 was dominant at a ratio of approximately 100:1 in the ocular infections. In contrast, the HSV-2 genome was present at a 4-40 times higher frequency in isolates from genital herpes patients. There was no obvious difference between the clinical course of mixed infection and those of single HSV-1 or HSV-2 infections. This study indicated that the frequency of mixed infection with both HSV-1 and HSV-2 is comparatively higher than those of previous reports. The genome ratio of HSV-1 and HSV-2 reflects the preference of each HSV type for the target organ.     

Oral bromovinyldeoxyuridine therapy for herpes simplex and varicella-zoster virus infections in severely immunosuppressed patients: a preliminary clinical trial

Twenty-five patients with haematological diseases were treated orally with the highly potent and selective anti-herpes agent, bromovinyldeoxyuridine (BVDU), in a dosage of 7.5 mg/kg/day (divided over three or four doses a day) for 5 days for an intercurrent mucocutaneous herpesvirus infection. Of these 25 patients, 8 were severely granulocytopenic at the time of the viral infection, and 12 recently had undergone bone-marrow transplantation; 5 were under cytotoxic therapy for a lymphoproliferative disorder; 13 had herpes simplex virus type 1 (HSV-1); 1 had herpes simplex virus type 2 (HSV-2); and 11 had varicella-zoster virus (VZV) infection. In all but two patients, BVDU arrested progression of the HSV or VZV infection within 1-2 days after treatment was started. One of the two patients who failed to respond to BVDU had an HSV-2 infection. The other had an HSV-1 infection, which was highly sensitive to BVDU in vitro; BVDU may have failed in this patient because of incomplete drug intake or profuse diarrhoea, or both. The results of this preliminary uncontrolled clinical trial suggest that BVDU may be an effective and safe drug for the oral treatment of HSV-1 and VZV infections in severely immunosuppressed patients.     

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.     

Herpes simplex virus infection of the placenta. The role of molecular pathology in the diagnosis of viral infection of placental-associated tissues.

Herpes simplex infections of the placenta and its associated tissues are rarely seen and, consequently, the pathologic features are not well understood. We describe the occurrence of herpes simplex virus infection of the placenta that was limited to maternal-derived cells of the subchorionic tissue. The microscopic findings were remarkable for both the absence of inflammation and characteristic viral inclusions. However, in situ DNA hybridization, as well as immunohistochemistry using antibody to herpes simplex virus type 2, revealed herpes virus infection of decidualized cells in the decidua capsularis. The significance of this finding, and the importance of molecular techniques for the diagnosis of herpes virus infection in placental pathology, are discussed.     

ELISA for the detection of herpes simplex virus antigens in the cerebrospinal fluid of patients with encephalitis

An inhibition enzyme-linked immunosorbent assay (ELISA) for the detection of herpes simplex virus antigens in cerebrospinal fluid (CSF) has been developed. A Triton X-100 extract of herpes simplex virus type 1 (HSV-1) infected HEp-2 cells was used to coat wells of polyvinyl chloride plates. Rabbit anti-HSV-1 globulin served as the reference antibody and the CSF specimens were tested at a final dilution of 1:4. Positive results were obtained in CSF specimens from 11/18 (61%) neonates with HSV infection, 15/23 (65%) older individuals with HSV culture positive brain biopsies, and in 4/29 (14%) patients with culture negative brain biopsies. The assay was negative with CSF from 14 infants without HSV infections, from 30 patients with bacterial meningitis and 10 with cryptococcal meningitis. The test was positive in 10/21 patients within 10 days of onset, 11/14 within 11-20 days, and in 5/6 more than 20 days after onset of the herpetic infection. The overall sensitivity of the assay was 63% and the specificity was 95%.     

Polymorphism of herpes simplex virus type 1 and 2 DNA from laboratory strains and clinical material from patient with genital herpes

Polymerase chain reaction (PCR), that can amplify a fragment of the DNA-polymerase gene of 4 herpes viruses, i.e. herpes simplex viruses, type 1 (HSV-1), herpes simplex viruses, type 2 (HSV-2), Epstein-Barr virus and cytomegalovirus, was made use of to study the genetic polymorphism of HSV-1 and HSV-2 strains. The obtained amplicons were analyzed by the method of restriction-size fragments' polymorphism (RSFP) with restrictases Rsal, Taql and Hinfl. Four HSV-1 strains had an identical restriction profile. Strain G (HSV-2) also displayed the expected restriction profiles, however, contradictory results were obtained for strain BH (HSV-2): the restriction profiles with restrictases Hinfl and Rsal corresponded to HSV-2, and the restriction profile with Taql corresponded to HSV-1. The sequencing of appropriate fragments of strains G and BH revealed a dot-type mutation localized in Taql restriction site. The thus worked out PCR was used jointly with RSFP in the genotyping of 75 urogenital samples obtained from women with genital herpes who were treated at Moscow patient-care facilities. HSV-1 and HSV-2 were detected in 18 (24%) and 57 (76%) of samples, respectively. No changes were registered in the restriction profile for HSV-2 among the investigated samples and all of them had the restriction profile similar to that of strain G. The conclusion is that genital herpes associated with HSV-2 is genetically stable within its Moscow population.     

Detection of herpes simplex virus DNA by in situ hybridization technique and polymerase chain reaction in Papanicolaou-stained cervicovaginal smears

Papanicolaou-stained cervicovaginal smears from six patients with herpes simplex virus (HSV) infection were destained and reprocessed by means of in situ hybridization (ISH) technique to demonstrate the presence of HSV DNA utilizing biotinylated probe. Positive results were obtained in all six cases with intense staining signal for the HSV DNA in the nuclei of cells having a ground-glass nuclear appearance as well as in multinucleated giant cells. Furthermore, a hybridization signal was also noted in smears that had been prepared as much as 3 yr previously. HSV type 2-specific antigen was confirmed in six destained smears by means of immunoperoxidase staining. Moreover, polymerase chain reaction (PCR) was also performed for four patients from Pap-destained cervicovaginal smears. Amplified HSV DNA was detected in all four cases as 106 basepair PCR products by polyacrylamide gel electrophoresis. The combined use of cytology and the ISH technique and PCR amplification was of great value for the rapid cytodiagnosis of genital infection of HSV.     

表达单纯疱疹病毒Ⅱ型糖蛋白D的重组痘苗病毒活疫苗株的建立

我们以前曾报道，表达单纯疱疹病毒Ⅱ型糖蛋白Ｄ（ＨＳＶ－２ｇＤ）的重组痘苗病毒（实验疫苗株）能保护被免疫小鼠抵抗致死量ＨＳＶ－２病毒的攻击。在此工作基础上，严格按人用疫苗研究要求的实验条件，成功地建立了表达ＨＳＶ－２ｇＤ的重组痘苗病毒活疫苗株。首先将经聚合酶链反应（ＰＣＲ）修饰的ＨＳＶ－２ｇＤ基因插入痘苗表达质粒ｐＪＳＢ１１７５，置于痘苗病毒Ｐ７５Ｋ早／晚期启动子控制下。将此重组质粒用Ｌｉｐｏｆｅｃｔｉｎ方法转染已受野型ＴＫ＋痘苗病毒天坛７６１株感染的人胚肺二倍体细胞。经同位素探针（３２Ｐ－ＨＳＶ－２ｇＤ）原位杂交法和３轮蚀斑纯化，筛选出基因组内整合有ＨＳＶ－２ｇＤ基因的重组痘苗病毒。斑点和Ｓｏｕｔｈｅｒｎ杂交证实，ＨＳＶ－２ｇＤ基因已插入痘苗病毒基因组内预期的ＴＫ区段，间接免疫荧光检测显示，重组病毒感染细胞后能有效地表达ＨＳＶ－２ｇＤ蛋白。     

表达单纯疱疹病毒Ⅱ型糖蛋白D的重组痘苗病毒活疫苗…

我们以前曾报道，表达单纯疱疹病毒Ⅱ型糖蛋白的重组痘苗病毒能保护被免疫小鼠抵抗致死量ＨＳＶ－２病毒的攻击。在此工作基础上，严格按人用疫苗研究要求的实验条件，成功地建立了表达ＨＳＶ－２ｇＤ的重组痘苗病毒活疫苗株，首先将经聚合酶链反应修饰的ＨＳＶ－２ｇＤ基因插入痘苗表达质粒ｐＪＳＢ１１７５，置于痘苗病毒Ｐ７．５Ｋ早／晚期启动子控制下，将同位重组质粒用Ｌｉｐｏｆｅｃｔｉｎ方法转染已受野型ＴＫ＾＋痘苗病毒天     

DNA polymerase in pseudorabies virus infected cells.

The DNA polymerase activity in BHK 21/C13 cells infected with pseudorabies virus is inhibited by incubation with antiserum to pseudorabies but not by incubation with pre-immune serum or by antiserum to herpes simplex virus type 1 (HSV-1) or herpes simplex virus type 2 (HSV-2). It also differs from the cell enzyme and the enzymes in HSV-1 or HSV-2 infected cells in its requirement for KCl in the in vitro assay. It seems likely, therefore, that pseudorabies virus specifies its own DNA polymerase.     

Distribution of herpes simplex virus types 1 and 2 genomes in human spinal ganglia studied by PCR and in situ hybridization

Clinical data indicate that the recurring herpes simplex virus (HSV) from oro-labial lesions is HSV subtype 1 and that the virus from genital lesions is HSV-2. This suggests that HSV-1 and HSV-2 reside in latent forms in the trigeminal ganglia and sacral ganglia, respectively. However, the distribution of latent HSV-1 and HSV-2 infections in human spinal ganglia has not been fully examined. This report concerns the application of polymerase chain reaction (PCR) and in situ hybridization (ISH) to such a study. By using PCR and employing the respective primers, HSV-1 and HSV-2 DNAs were detected in 207 of 524 samples from 262 spinal ganglia (from the cervical to the sacral ganglia) examined on both sides. The percentages of HSV-1 and HSV-2 detected in a given set of ganglia were similar, indicating an absence of site preference. By ISH, few but positive hybridization signals were detected evenly in sacral ganglia sections. The data suggest that regional specificity of recurrent HSV infections is not due to regional distribution of latent virus, but that local host factors may be important for recurrences. J. Med. Virol. 52:136–142, 1997. © 1997 Wiley-Liss, Inc.     

Detection of herpes simplex virus type 1 in herpetic ocular diseases by DNA-DNA hybridization using a biotinylated DNA probe

A diagnostic hybridization assay for detecting herpes simplex virus type 1 (HSV-1) in ocular specimens was developed using cloned viral DNA as a probe. This hybridization assay is based on visualizing a biotinylated probe that is hybridized to the target DNA by a streptavidin/alkaline phosphatase system. The time required for performing this assay system is only two days. This assay system could detect a probe which had been hybridized to as little as 1 pg of homologous DNA and did not cross-react with DNA of other human herpes viruses except that of herpes simplex virus type 2 (HSV-2) which showed weak cross-reactivity. The assay system was applied to experimental keratitis in albino rabbits and clinical specimens. In experimental keratitis in rabbits it was possible to detect HSV-1 DNA in the eye swab samples at least until the ninth day after virus inoculation. Five clinical specimens collected from patients with corneal ulcer or blepharitis contained HSV-1 DNA in spite of the failure of demonstration of viral antigen and/or virus isolation in two cases.     

Correlation of viral factors with cervical cancer in Taiwan.

The correlation of viral factors with cervical cancer was investigated. 27 cervical cancer biopsies and 29 normal cervical scrapings were determined by polymerase chain reaction method for 6 viruses, including human papillomavirus (HPV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus (HSV)-1, HSV-2, and human herpes virus (HHV)-8. Among 27 biopsies of cervical cancer, HPV was identified in 18. Of these HPV-positive specimens, 9 cases of HPV type 16 were identified, 2 cases of HPV type 18 and 1 case of mixed infection with HPV types 16 and 18 were identified. Among the HPV types detected, type-16 is the most closely associated with cervical cancer and type-18 ranks second. Of the remaining 6 cases, 1 case of HPV-45, 1 case of mixed infection with HPV type 35, CMV and HSV-2, and 4 cases of unidentified HPV type were also found. EBV, HSV-1 and HHV-8 were not found in the cervical cancer samples and might have no or little relationship with cervical cancer. Among the 29 specimens in the normal female control group, no viral infection was detected. The correlation of HPV with cervical cancer was significantly different between frozen tissues and paraffin-embedded tissues. Other viruses such as HSV-2 and CMV are not predictive of cervical cancer. They might not be involved in the oncogenic processes directly but might enhance the possibility of oncogenesis or infect cancer tissues opportunistically.     

Parallel detection of five human herpes virus DNAs by a set of real-time polymerase chain reactions in a single run.

BACKGROUND: Human herpes viruses cause a spectrum of diseases that are usually self-limiting but can be reactive during immuno-suppression and may then lead to severe or even life-threatening diseases. The LightCycler technology allows rapid polymerase chain reaction (PCR) including product analysis within a closed system. This approach has been demonstrated to be suitable for routine diagnostic virus detection. Several LightCycler PCR assays have been established to the detection of human herpes viruses. The assays vary in their detection formats and PCR cycling protocols. So, they cannot be performed within a single LightCycler run. OBJECTIVES: Development of four LightCycler PCR assays for parallel detection of DNA derived from human cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), and varicella-zoster virus (VZV) in a single run. STUDY DESIGN: Primers and hybridization probes were tailored to suit one LightCycler PCR program. LightCycler PCRs were established, detection limits were determined, and clinical samples were evaluated. RESULTS: With quantified herpes virus type specific DNA spiked into cerebrospinal fluid, serum or EDTA plasma the detection limits were found either at 500 or 250 viral DNA copies per ml depending on the virus DNA specific PCR and on the specimen type used. The applicability of the new LightCycler assays for routine molecular testing was evaluated by testing 96 clinical samples. CONCLUSION: The developed set of LightCycler PCRs permits parallel detection of CMV, EBV, HSV-1, HSV-2, and VZV in a single LightCycler run. The new molecular assays can easily be used to the rapid, simple, and convenient detection of herpes virus DNA in cerebrospinal fluid, serum and EDTA plasma in the routine diagnostic laboratory.     

Chronic meningitis due to herpes simplex virus in an immunocompetent host

Herpes simplex virus (HSV) typically causes mucocutaneous disease, encephalitis, and acute meningitis. There have been no previous reports of chronic meningitis due to this virus. A case of chronic meningitis due to herpes simplex virus type 2 (HSV-2) in a previously healthy 35-year-old woman whose predominant symptoms were headache and meningism without fever is described. Analysis of cerebrospinal fluid (CSF) revealed a lymphocytic pleocytosis, elevated protein, and hypoglycorrhachia. The diagnosis of herpes simplex meningitis was supported by the detection of HSV-2 DMA in CSF by polymerase chain reaction and by intrathecal production of HSV-specific antibody. The patient recovered after treatment with intravenous acyclovir and glucocorticoids.     

Herpes simplex virus type 1 infection associated with atrial myxoma

Some findings suggest an infectious factor in cardiac myxoma and certain histopathological features indicate herpes simplex virus type 1 (HSV-1) infection. We hypothesized that HSV-1 may be involved in the pathogenesis of cardiac myxoma. Paraffin-embedded tissue samples from 17 patients with atrial myxoma were investigated for HSV-1 antigen by immunohistochemistry and viral genomic DNA by nested polymerase chain reaction. The histogenesis and oncogenesis of atrial myxoma were assessed by the expression of calretinin, Ki67, and p53 protein, respectively. Autopsy myocardial samples, including endocardium from 12 patients who died by accident or other conditions, were used for comparison. HSV-1 antigen was detected in atrial myxoma from 12 of 17 patients: 8 of these 12 samples were positive also for HSV-1 DNA. No HSV-1 antigen or DNA was found in tissue from the comparison group. Antigens of HSV-2, varicella-zoster virus, Epstein-Barr virus, and cytomegalovirus were not found in atrial myxoma. Calretinin was found in myxoma cells of all 17 cases but Ki67 was present only in smooth muscle cells or infiltrating cells in some cases. p53 was not detectable in any myxoma. Most infiltrating cells were cytotoxic T lymphocytes. These data suggest that HSV-1 infection is associated with some cases of sporadic atrial myxoma and that these may result from a chronic inflammatory lesion of endocardium.     

Detection of viral DNA and activation of latent herpes simplex virus in the rabbit neural tissue

Both trigeminal ganglia, brain stem, and cornea from rabbits with established latent herpes simplex virus type 1 (HSV-1) infection were examined by explantation and by spot blot hybridization using strain 17 Kpn I fragments i, d, and h and the DNA extracted from above mentioned tissues. Correlation between positive hybridization and reactivation of infectious virus in the cultured explants was documented by enhanced hybridization with the DNA extracts from explanted ganglion samples. In addition, we found positive hybridization in some noncultured ganglion and brain stem samples which did not yield infectious virus by explantation. Keeping in mind the pitfalls of false positive hybridization, the results may indicate during latency the presence in neural tissues of HSV DNA sequences which did not spontaneously reactivate in culture.     

Recurrent herpes simplex virus type 1 infection precipitated by the impaired production of interleukin-2, alpha-interferon, and cell-mediated cytotoxicity

In order to understand whether immunosuppression due to the primary host defense defect provokes the reactivation of herpes simplex virus type 1 (HSV-1), 11 healthy persons were evaluated immunologically after recurrence of herpes. A decrease was found in the production of interleukin-2 (IL-2) and alpha-interferon (IFN-alpha) to HSV-1 antigen stimulation in the recrudescent stage (0 to 3 days after onset of the lesion). The cell-mediated cytotoxicity also decreased. To clarify the relationship between the immunosuppression and the occurrence of herpes recurrence, a further longitudinal study was undertaken measuring IL-2, IFN-alpha production, and cell mediated cytotoxicity every other week in 12 persons who suffered frequently from recurrent HSV-1 infection. IL-2 and IFN-alpha production after HSV-1 antigen stimulation and cell-mediated cytotoxicity before recurrence of herpes were lower than those without recurrence. These defective immunologic responses were persistent and became more pronounced during the recrudescent phase. The results suggest that the defect in the immunologic responses to HSV-1 antigen is related to the recurrence of herpes simplex.     

Amplification and characterization of herpesvirus DNA in cerebrospinal fluid from patients with acute encephalitis.

A single pair of oligonucleotide primers selected within a highly conserved region of the DNA polymerase gene of the herpesviruses was designed to amplify related viral genomes, i.e., herpes simplex virus type 1, herpes simplex virus type 2, Epstein-Barr virus, and cytomegalovirus, by the polymerase chain reaction. A simple restriction enzyme analysis of these amplified products allowed accurate characterization of the herpesvirus type. Ninety-nine cerebrospinal fluid samples from 36 patients (including newborns, children, and adults) with acute encephalitis were tested for the presence and identification of herpesvirus DNA by this approach. High levels of viral DNA, which were readily visualized by simple ethidium bromide staining, were found in all these patients from the first days of the disease and, in some cases, until the third week following the onset of acute encephalitis. The herpesvirus type was rapidly identified by enzymatic digestion in 33 patients' samples and by hybridization and direct sequencing in the last 3 patients' samples. Our results show that the polymerase chain reaction provides a highly sensitive and specific technique for the identification of herpesviruses DNA in cerebrospinal fluid that should be of value for early and rapid diagnosis, therapeutic decisions, prognostic evaluation, and epidemiological studies.