人类疱疹病毒8型包膜糖蛋白K8.1单克隆抗体的制备与鉴定

目的：制备人类疱疹病毒8型（HHV-8）包膜糖蛋白K8．1的单克隆抗体，并对其特异性进行鉴定。方法：用异丙基硫代一β—D一半乳糖苷（IPTG）诱导含重组质粒pGEX-6p-1 ＋K8．1的大肠杆菌BL21表达符胱甘肽-S-转移酶GST／K8．1融合蛋白，将以包涵体形式存在的融合蛋白进行变性、复性、复性后的Glutathione Sepharose 4B亲和层析柱纯化。以纯化的GST／K8．1融合蛋白为抗原免疫BALB／c小鼠．常规杂交瘤技术制备单克隆抗体。采用酶联免疫吸附试验（ELISA）筛选分泌K8．1单抗的杂交瘤细胞株。免疫组化染色（IHC）和Western blot法鉴定单抗的特异性。结果：建立了一株稳定分泌抗K8．1单抗的杂交瘤细胞株．命名为3G10：IHC和Westerfl blot法显示．3G10株单抗能特异地识别HHV-8K8．1蛋白。结论：成功制备出抗HHV-8包膜糖蛋白K8．1的单克隆抗体。     

Integration of human herpesvirus 6 in a Burkitt's lymphoma cell line

Human herpesvirus 6 (HHV-6) genome has been found in several human lymphoid malignancies, but configuration of the HHV-6 genome has not been well delineated. We established the HHV-6-positive, Epstein-Barr virus-negative Burkitt's lymphoma cell line Katata. In this study we investigated the status of the HHV-6 genome in Katata cells. Neither linear nor circular HHV-6 DNA was detected by Gardella gel analysis. The fluorescence in situ hybridization technique enabled us to directly visualize the integrated HHV-6 DNA at the single-cell level. Only one integrated site of viral DNA was detected in metaphase chromosomes and it was preferentially located at the long arm of chromosome 22 (22q13). Treatment of the cells with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or with calcium ionophore A23187 led to induction of the HHV-6 immediate-early gene as well as the late gene. Sodium n-butyrate also gave rise to expression of the HHV-6 genes. The TPA inducibility was synergistically enhanced when combined with A23187 or n-butyrate. Our study provides, for the first time, an in vitro model system of latent HHV-6 infection whose genome is integrated into host DNA of lymphoma cells.     

The Acute Retinal Necrosis Syndrome: Part 2: Histopathology and Etiology

The acute retinal necrosis syndrome is manifested by diffuse uveitis, vitritis, retinal vasculitis, and acute necrotizing retinitis (see Part 1). We studied the histopathology and electron microscopic findings of an eye enucleated from a 67-year-old man with typical acute retinal necrosis. Histology showed profound acute necrosis of the retina, retinal arteritis, and eosinophilic intranuclear inclusions in retinal cells. Electron microscopy demonstrated a herpes group virus in all layers of affected retina. The implications of these findings for antiviral and other treatments are discussed.     

The Ozobranchus leech is a candidate mechanical vector for the fibropapilloma-associated turtle herpesvirus found latently infecting skin tumors on Hawaiian green turtles (Chelonia mydas)

Fibropapillomatosis (FP) of marine turtles is a neoplastic disease of ecological concern. A fibropapilloma-associated turtle herpesvirus (FPTHV) is consistently present, usually at loads exceeding one virus copy per tumor cell. DNA from an array of parasites of green turtles (Chelonia mydas) was examined with quantitative PCR (qPCR) to determine whether any carried viral loads are sufficient to implicate them as vectors for FPTHV. Marine leeches (Ozobranchus spp.) were found to carry high viral DNA loads; some samples approached 10 million copies per leech. Isopycnic sucrose density gradient/qPCR analysis confirmed that some of these copies were associated with particles of the density of enveloped viruses. The data implicate the marine leech Ozobranchus as a mechanical vector for FPTHV. Quantitative RT-PCR analysis of FPTHV gene expression indicated that most of the FPTHV copies in a fibropapilloma have restricted DNA polymerase expression, suggestive of latent infection.     

Highly selective escape from KSHV-mediated host mRNA shutoff and its implications for viral pathogenesis

During Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) lytic infection, many virus-encoded signaling molecules (e.g., viral G protein-coupled receptor [vGPCR]) are produced that can induce host gene expression in transiently transfected cells, and roles for such induced host genes have been posited in KS pathogenesis. However, we have recently found that host gene expression is strongly inhibited by 10-12 h after lytic reactivation of KSHV, raising the question of whether and to what extent de novo host gene expression induced by viral signaling molecules can proceed during the lytic cycle. Here, we show by microarray analysis that expression of most vGPCR target genes is drastically curtailed by this host shutoff. However, rare cellular genes can escape the host shutoff and are potently up-regulated during lytic KSHV growth. Prominent among these is human interleukin-6, whose striking induction may contribute to the overexpression of this cytokine in several disease states linked to KSHV infection.     

Human herpesviruses 6 and 7 and central nervous system infection in children

The role and frequency of human herpesviruses (HHV)-6 and -7 in central nervous system (CNS) diseases of children are unclear. Cerebrospinal fluid samples from 245 pediatric patients (median age 43 days), submitted for evaluations of possible sepsis or of neurologic symptoms, were tested for HHV-6 and HHV-7 DNA by polymerase chain reaction. HHV-6 DNA was found in 3 of 245 samples, and HHV-7 was found in 0 of 245 samples. The three patients with HHV-6 DNA were <2 months of age. HHV-6 was likely pathogenic in two patients with meningitis who lacked evidence of another microbiologic cause. HHV-6 and HHV-7 are uncommon causes of CNS infection in children. HHV-6 may occasionally cause meningitis in young infants.     

Human herpesvirus type 8 infections among solid organ transplant recipients

Human herpes virus 8 (HHV-8) is known to be associated with Kaposi's sarcoma (KS), primary effusion lymphoma (PEL) and a form of Castleman's disease. Recently, it has also been shown to be associated with acute bone marrow failure in transplant patients. While, the full spectrum of clinical manifestations due to HHV-8 is yet to be defined in transplant recipients, it is known to cause post-transplant KS as a result of primary as well as secondary infection. This review will discuss the possible role of HHV-8 as a cause of disease in solid organ transplant recipients by focussing on important issues, including the biology of the virus, epidemiology, clinical manifestations, laboratory diagnosis and treatment, followed by a discussion of issues of relevance to the pediatric transplant recipient.     

Human herpesvirus 8 and Epstein-Barr virus-related monotypic large B-cell lymphoproliferative disorder coexisting with mixed variant of Castleman's disease in a lymph node of a renal transplant recipient

Human herpesvirus 8 (HHV8) has been related to some malignant lymphoproliferations, including post-transplant lymphoproliferative disorders (PTLD). We describe a case of a HHV8 and Epstein-Barr virus (EBV) positive large B-cell lymphoproliferation coexisting with Castleman's disease in the same lymph node of a long-term renal transplant recipient. Biopsy revealed mixed type of Castleman's disease and anaplastic cells showing IgA restriction, although molecular analysis failed to detect monoclonality. Only large cells were co-infected by both EBV and HHV8. After reduction of immunosuppression, the lesion partially regressed. After 1 yr, local evolution required surgery followed by irradiation. The present case represents a unique form of localized monotypic but polyclonal large cell PTLD associated with Castleman's disease. It can be added to PTLD with HHV8 and EBV co-infection.     

Human herpesvirus 6 in the pathogenesis of multiple sclerosis

Multiple sclerosis (MS) is one of the most common disabling neurological diseases affecting young adults. It is a chronic disease characterised by inflammation and demyelination. The aetiology of MS is still unknown, but involvement of viruses has been suspected for many years. Recently much interest has focused on human herpesvirus 6 (HHV-6), since the virus has been detected in MS plaques in the brain and patients with MS have been shown to have an aberrant immune response to HHV-6. Results from different studies are, however, conflicting and in the light of the long list of previous claims to have found the viral aetiology of MS it is necessary to interpret the HHV-6 findings with great caution. Possible mechanisms for virally induced demyelination and autoimmunity are discussed in this review, and the evidence for and against a role for HHV-6 in MS is summarised.