RESUMEN
Human herpes simplex virus type 1 (HSV-1) and varicella-zoster virus (VZV) were isolated in the bilateral trigeminal ganglia of 12 human cadavers with no history of herpes-related symptoms within 1-5 days of death. Sixteen trigeminal ganglia were subjected to explant culture by using Vero cells, but no cytopathogenic effects (CPE) were observed. However, when another eight trigeminal ganglia were placed in a cell strainer and kept from direct contact with Vero cells during culture, CPE were clearly apparent in all cultures. The amount of DNA in the culture supernatants of 16 trigeminal ganglia decreased over time; 12 and 9 of these samples were PCR-positive for HSV-1 and VZV, respectively. In new Vero cells inoculated with supernatants collected 2 days after culture initiation, immunofluorescence staining revealed HSV-1 and VZV in 6 and 5 of 8 trigeminal ganglia, respectively. HSV-1 and VZV DNA was detected in supernatants collected 3 and 7 days after culture initiation and in Vero cells collected after culture completion, but real-time PCR revealed the DNA amounts decreased over time. There was less VZV DNA than HSV-1 DNA. These results demonstrate that infective HSV-1 and VZV can be isolated in culture, and confirm that viable HSV-1 and VZV persist in human trigeminal ganglia for some time after death.
Asunto(s)
Cadáver , ADN Viral/aislamiento & purificación , Herpesvirus Humano 1/aislamiento & purificación , Herpesvirus Humano 3/aislamiento & purificación , Ganglio del Trigémino/virología , Anciano , Anciano de 80 o más Años , Animales , Chlorocebus aethiops , ADN Viral/genética , Femenino , Herpesvirus Humano 1/genética , Herpesvirus Humano 3/genética , Humanos , Masculino , Viabilidad Microbiana , Persona de Mediana Edad , Técnicas de Cultivo de Órganos , Células Vero , Carga ViralRESUMEN
Cytomegalovirus (CMV) retinitis is characterized by alterations in retinal cell function and host responses to virus replication. The goal of this study was to evaluate the induction of cyclooxygenase-2 (COX-2) and prostaglandin (PGE) in CMV infected human retinal pigment epithelial (RPE) cells and to determine their effect on virus replication. CMV immediate early (IE) protein and COX-2 proteins were identified in RPE cells in retinal tissue sections from patients with CMV retinitis. COX-2 mRNA and protein were induced after CMV infection of human RPE cell cultures. CMV infection of RPE cells induced translocation of NF-kappaB from the cytoplasm to the nucleus. PGE1 and PGE2 were significantly (p<0.001) increased in human RPE cell cultures infected with CMV. Inhibition of CMV IE gene by antisense oligonucleotides abrogated induction of mRNA for COX-2 and protein synthesis of COX-2 and PGE2. PGE enhanced CMV plaque formation and real time PCR analysis revealed that PGE treatment significantly increased CMV DNA copy numbers. These studies demonstrate that when CMV replicates within human RPE cells, COX-2 induction augments virus replication via the PGE pathway. The induction of COX-2 and PGE during retinal CMV infection may augment virus replication and alter a variety of retinal physiological responses.
Asunto(s)
Ciclooxigenasa 2/biosíntesis , Citomegalovirus/fisiología , Epitelio Pigmentado Ocular/virología , Prostaglandinas/biosíntesis , Replicación Viral , Núcleo Celular/química , Células Cultivadas , Ciclooxigenasa 2/genética , Citomegalovirus/crecimiento & desarrollo , Retinitis por Citomegalovirus/patología , Retinitis por Citomegalovirus/virología , Citoplasma/química , ADN Viral/análisis , Expresión Génica , Humanos , Proteínas Inmediatas-Precoces/análisis , Proteínas Inmediatas-Precoces/antagonistas & inhibidores , Inmunohistoquímica , Microscopía Fluorescente , FN-kappa B/metabolismo , Oligonucleótidos Antisentido/genética , ARN Mensajero/análisis , ARN Mensajero/genética , Retina/química , Ensayo de Placa Viral , Proteínas Virales/análisisRESUMEN
PURPOSE: To evaluate the effects of human cytomegalovirus (HCMV) infection on chemokine gene expression and secretion by human retinal pigment epithelial (HRPE) cell cultures. METHODS: HRPE cells were infected with HCMV (strain AD169) at an MOI of 5. Culture supernatants, collected at various postinoculation days, were used for the analyses of chemokines by ELISA. The steady state levels of chemokine and chemokine receptor mRNA were analyzed by RT-PCR. Effects of interferon and MCP-1 on HCMV replication in HRPE cells were evaluated by plaque assays. RESULTS: HRPE cells infected with HCMV exhibited characteristic cytopathic effects. The reduction in the levels of monocyte chemotactic protein (MCP)-1 and -3 mRNA in HCMV-infected HRPE cells was observed in comparison to uninfected HRPE cells. In contrast, HCMV infection enhanced IL-8 mRNA levels, whereas regulated on activation normal T-cell expressed and secreted (RANTES) mRNA was not detectable in either control or infected HRPE cells. A significant decrease in MCP-1 (P < 0.01) and MCP-3 (P < 0.05), but a significant increase in IL-8 (P < 0.05), protein secretion was observed. Expression of the chemokine receptors CCR2, specific for MCP-1, and CXCR1 and CXCR2, specific for IL-8, were not altered by HCMV infection. Treatment of HRPE cultures with MCP-1 had no significant effect on HCMV replication in HRPE cells. CONCLUSIONS: HCMV infection in HRPE cells resulted in the modulation of MCP-1, MCP-3, and IL-8. Because chemokines facilitate the activation of leukocytes and their migration to the sites of inflammation, the modulation of chemokine production by the virus suggests a role for chemokines in immune evasion and/or immunopathogenesis of CMV retinitis.