Recombinant feline leukemia virus (FeLV) variants establish a limited infection with altered cell tropism in specific-pathogen-free cats in the absence of FeLV subgroup A helper virus.
(17/2725)
Feline leukemia virus subgroup B (FeLV-B) is commonly associated with feline lymphosarcoma and arises through recombination between endogenous retroviral elements inherited in the cat genome and corresponding regions of the envelope (env) gene from FeLV subgroup A (FeLV-A). In vivo infectivity for FeLV-B is thought to be inefficient in the absence of FeLV-A. Proposed FeLV-A helper functions include enhanced replication efficiency, immune evasion, and replication rescue for defective FeLV-B virions. In vitro analysis of the recombinant FeLV-B-like viruses (rFeLVs) employed in this study confirmed these viruses were replication competent prior to their use in an in vivo study without FeLV-A helper virus. Eight specific-pathogen-free kittens were inoculated with the rFeLVs alone. Subsequent hematology and histology results were within normal limits, however, in the absence of detectable viremia, virus expression, or significant seroconversion, rFeLV proviral DNA was detected in bone marrow tissue of 4/4 (100%) cats at 45 weeks postinoculation (pi), indicating these rFeLVs established a limited but persistent infection in the absence of FeLV-A. Altered cell tropism was also noted. Focal infection was seen in T-cell areas of the splenic follicles in 3/4 (75%) rFeLV-infected cats analyzed, while an FeLV-A-infected cat showed focal infection in B-cell areas of the splenic follicles. Nucleotide sequence analysis of the surface glycoprotein portion of the rFeLV env gene amplified from bone marrow tissue collected at 45 weeks pi showed no sequence alterations from the original rFeLV inocula. (+info)
Gene transfer to human pancreatic endocrine cells using viral vectors.
(18/2725)
We have studied the factors that influence the efficiency of infection of human fetal and adult pancreatic endocrine cells with adenovirus, murine retrovirus, and lentivirus vectors all expressing the green fluorescent protein (Ad-GFP, MLV-GFP, and Lenti-GFP, respectively). Adenoviral but not retroviral vectors efficiently infected intact pancreatic islets and fetal islet-like cell clusters (ICCs) in suspension. When islets and ICCs were plated in monolayer culture, infection efficiency with all three viral vectors increased. Ad-GFP infected 90-95% of the cells, whereas infection with MLV-GFP and Lenti-GFP increased only slightly. Both exposure to hepatocyte growth factor/scatter factor (HGF/SF) and dispersion of the cells by removal from the culture dish and replating had substantial positive effects on the efficiency of infection with retroviral vectors. Studies of virus entry and cell replication revealed that cell dispersion and stimulation by HGF/SF may be acting through both mechanisms to increase the efficiency of retrovirus-mediated gene transfer. Although HGF/SF and cell dispersion increased the efficiency of infection with MLV-GFP, only rare cells with weak staining for insulin were infected, whereas approximately 25% of beta-cells were infected with Lenti-GFP. We conclude that adenovirus is the most potent vector for ex vivo overexpression of foreign genes in adult endocrine pancreatic cells and is the best vector for applications where high-level but transient expression is desired. Under the optimal conditions of cell dispersion plus HGF/SF, infection with MLV and lentiviral vectors is reasonably efficient and stable, but only lentiviral vectors efficiently infect pancreatic beta-cells. (+info)
Detection of Epstein-Barr virus (EBV) in hepatocellular carcinoma tissue: a novel EBV latency characterized by the absence of EBV-encoded small RNA expression.
(19/2725)
In this study, we investigated the presence of Epstein-Barr virus (EBV) in liver tissue from 35 patients with hepatocellular carcinoma (HCC). EBV DNA was detected in 13 patients (37%) by Southern blot hybridization. In 10 of these patients, EBV DNA was present in tumor tissue only, whereas in the other 3, it was detected in both tumor and nontumor tissues. The quantity of EBV DNA detected was equivalent to 1-10 viral DNA molecules/100 cells. EBV-determined nuclear antigen was detected in 7-13% of the carcinoma cells in three tumor tissue samples that contained approximately one copy of the EBV genome/10 cells. A single terminal fragment of EBV DNA was identified in these tissues, suggesting that the EBV-infected cells in HCC represent clonal proliferation. Western blotting and reverse transcription-polymerase chain reaction analyses demonstrated that these three tumor tissue specimens were positive for EBV-determined nuclear antigen 1 and BamHI A transcripts but were negative for the other latent EBV products, including EBV-encoded small RNA. The results indicated that there is a high EBV load in HCC tissue and that all of the HCC tissue examined showed a novel pattern of EBV latency characterized by absence of EBV-encoded small RNA expression. (+info)
Expression, purification and immunological characterization of the transforming protein E7, from cervical cancer-associated human papillomavirus type 16.
(20/2725)
E7 is the major oncogenic protein produced in cervical cancer-associated human papillomavirus type 16 (HPV16). This protein was expressed in Escherichia coli as a glutathione-S-transferase (GST) fusion protein. E7-enriched inclusion bodies were collected from bacterial lysates, were solubilized in 10 M urea, and the protein was purified using anion exchange column chromatography. After removal of endotoxin with serial Triton X-114 extractions, material of high purity (about 90%) was obtained, which is suitable for use in a human clinical trial. This material was immunogenic, and when used as a vaccine, protected mice against challenge with an HPV16 E7 DNA transfected tumour cell line. Based on this observation, the E7GST fusion protein is currently being used in a human clinical trial of a vaccine against HPV16-induced cervical cancer. This fusion protein could be cleaved with thrombin to remove the GST fusion part and further purified by preparative SDS gel electrophoresis to obtain free E7 with > 98% purity. (+info)
Differential chemokine expression in tissues involved by Hodgkin's disease: direct correlation of eotaxin expression and tissue eosinophilia.
(21/2725)
Hodgkin's disease (HD) is a lymphoid malignancy characterized by infrequent malignant cells surrounded by abundant inflammatory cells. In this study, we examined the potential contribution of chemokines to inflammatory cell recruitment in different subtypes of HD. Chemokines are small proteins that are active as chemoattractants and regulators of cell activation. We found that HD tissues generally express higher levels of interferon-gamma-inducible protein-10 (IP-10), Mig, RANTES, macrophage inflammatory protein-1alpha (MIP-1alpha), and eotaxin, but not macrophage-derived chemotactic factor (MDC), than tissues from lymphoid hyperplasia (LH). Within HD subtypes, expression of IP-10 and Mig was highest in the mixed cellularity (MC) subtype, whereas expression of eotaxin and MDC was highest in the nodular sclerosis (NS) subtype. A significant direct correlation was detected between evidence of Epstein-Barr virus (EBV) infection in the neoplastic cells and levels of expression of IP-10, RANTES, and MIP-1alpha. Levels of eotaxin expression correlated directly with the extent of tissue eosinophilia. By immunohistochemistry, IP-10, Mig, and eotaxin proteins localized in the malignant Reed-Sternberg (RS) cells and their variants, and to some surrounding inflammatory cells. Eotaxin was also detected in fibroblasts and smooth muscle cells of vessels. These results provide evidence of high level chemokine expression in HD tissues and suggest that chemokines may play an important role in the recruitment of inflammatory cell infiltrates into tissues involved by HD. (+info)
Protection against establishment of retroviral persistence by vaccination with a live attenuated virus.
(22/2725)
Many human viruses not only cause acute diseases but also establish persistent infections. Such persistent viruses can cause chronic diseases or can reactivate to cause acute diseases in AIDS patients or patients receiving immunosuppressive therapies. While the prevention of persistent infections is an important consideration in the design of modern vaccines, surprisingly little is known about this aspect of protection. In the current study, we tested the feasibility of vaccine prevention of retroviral persistence by using a Friend virus model that we recently developed. In this model, persistent virus can be detected at very low levels by immunosuppressing the host to reactivate virus or by transferring persistently infected spleen cells into highly susceptible mice. Two vaccines were analyzed, a recombinant vaccinia virus vector expressing Friend virus envelope protein and a live attenuated Friend virus. Both vaccines reduced pathogenic virus loads to levels undetectable by infectious center assays. However, only the live, attenuated vaccine prevented immunosuppression-induced reactivation of persistent virus. Thus, even very low levels of persistent Friend virus posed a significant threat during immunosuppression. Our results demonstrate that vaccine protection against establishment of retroviral persistence is attainable. (+info)
Complete sequence of enzootic nasal tumor virus, a retrovirus associated with transmissible intranasal tumors of sheep.
(23/2725)
The sequence of the complete genome of ovine enzootic nasal tumor virus, an exogenous retrovirus associated exclusively with contagious intranasal tumors of sheep, was determined. The genome is 7,434 nucleotides long and exhibits a genetic organization characteristic of type B and D oncoviruses. Enzootic nasal tumor virus is closely related to the Jaagsiekte sheep retrovirus and to sheep endogenous retroviruses. (+info)
Jaagsiekte retrovirus is widely distributed both in T and B lymphocytes and in mononuclear phagocytes of sheep with naturally and experimentally acquired pulmonary adenomatosis.
(24/2725)
Jaagsiekte sheep retrovirus (JSRV) is a type D retrovirus specifically associated with a contagious lung tumor of sheep, sheep pulmonary adenomatosis (SPA). JSRV replicates actively in the transformed epithelial cells of the lung, and JSRV DNA and RNA have been detected in lymphoid tissues of naturally affected animals. To determine the lymphoid target cells of JSRV, CD4(+) T cells, CD8(+) T cells, B lymphocytes, and adherent cell (macrophage/monocyte) populations were isolated from the mediastinal lymph nodes of naturally affected sheep and lambs inoculated with JSRV. Cells were enriched to high purity and then analyzed for JSRV proviral DNA by heminested PCR, and the proviral burden was quantitated by limiting dilution analysis. JSRV proviral DNA was found in all subsets examined but not in appropriate negative controls. In sheep naturally affected with SPA, JSRV proviral burden was greatest in the adherent cell population. In the nonadherent lymphocyte population, surface immunoglobulin-positive B cells contained the greatest proviral burden, while CD4(+) and CD8(+) T cells contained the lowest levels of JSRV proviral DNA. In most of the cases (5 of 8), provirus also could be detected in the peripheral blood mononuclear cell (PBMC) population. A kinetic study of JSRV infection in the mediastinal lymphocyte population of newborn lambs inoculated with JSRV found that JSRV proviral DNA could be detected as early as 7 days postinoculation before the onset of pulmonary adenomatosis, although the proviral burden was greatly reduced compared to adult natural cases. This was reflected in the levels found in PBMC since proviral DNA was detected in 2 of 13 animals. At the early time points studied (7 to 28 days postinoculation) no one subset was preferentially infected. These data indicate that JSRV can infect lymphoid and phagocytic mononuclear cells of sheep and that dissemination precedes tumor formation. Infection of lymphoid tissue, therefore, may play an important role in the pathogenesis of SPA. (+info)