RESUMEN
The early stages of Venezuelan equine encephalitis virus (VEE) pathogenesis in the mouse model have been examined using a genetic approach. Disease progression of a molecularly cloned single-site mutant was compared with that of the parental virus to determine the step in the VEE pathogenetic sequence at which the mutant was blocked. Assuming that such a block constitutes a genetic screen, isolates from different tissues thought to be distal to the block in the VEE pathogenetic sequence were analyzed to determine the pathogenetic step at which revertants of the mutant were selected. Directed mutation and analysis of reversion in vivo provide two powerful genetic tools for the dissection of the wild-type VEE pathogenetic sequence. Virus from the parental virulent clone, V3000, first replicated in the draining lymph node after subcutaneous inoculation in the left rear footpad. Movement of a cloned avirulent mutant, V3010 (E2 76 Glu to Lys), to the draining lymph node was impaired, replication in the node was delayed, and spread beyond the draining lymph node was sporadic. Serum, contralateral lymph node, spleen, and brain isolates from V3010 inoculated animals were invariably revertant with respect to sequence at E2 76 and/or virulence in mice. Revertants isolated from serum and contralateral lymph node retained the V3010 E2 Lys 76 mutation but also contained a second-site mutation, Glu to Lys at E2 116. Modification of the V3010 clone by addition of the second-site mutation at E2 116 produced a virus that bypassed the V3010 block at the draining lymph node but that did not possess full wild-type capacity for replication in the central nervous system or for induction of mortality. A control construct containing only the E2 116 reverting mutation on the V3000 background was identical to V3000 in terms of early pathogenetic steps and virulence. Therefore, analysis of mutant replication and reversion in vivo suggested (1) that the earliest steps in VEE pathogenesis are transit to the draining lymph node and replication at that site, (2) that the mutation in V3010 impairs transit to the draining lymph node and blocks dissemination to other tissues, and (3) that reversion can overcome the block without restoring full virulence.
Asunto(s)
Virus de la Encefalitis Equina Venezolana/genética , Virus de la Encefalitis Equina Venezolana/patogenicidad , Encefalomielitis Equina Venezolana/virología , Mutación Puntual/genética , Supresión Genética/genética , Animales , Encéfalo/virología , Línea Celular , Clonación Molecular , Progresión de la Enfermedad , Virus de la Encefalitis Equina Venezolana/fisiología , Encefalomielitis Equina Venezolana/mortalidad , Femenino , Ganglios Linfáticos/virología , Ratones , Fenotipo , ARN Viral/genética , ARN Viral/metabolismo , Bazo/virología , Relación Estructura-Actividad , Vacunas Atenuadas/genética , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/fisiología , Vacunas Virales/genética , Viremia , Virulencia/genética , Replicación ViralRESUMEN
Home care work in metropolitan areas is a source of employment for immigrant women of color. Service work of this type intertwines domestic and caring labor in ways that reinforce the historically gendered and racialized nature of the work. Such macro level economic and political issues are played out at the micro level of daily service provided within elderly clients' homes. A study of these processes in home care work was carried out in urban southern Ontario in two nonprofit home care agencies. In-depth interviews and focus groups held with visible minority home care workers suggested that workers deal daily with racist attitudes and behaviors from clients and their families; agencies recognize these oppressive processes but usually handle them on a case-by-case basis through supervisors; and home care workers handle racism on the job as they do in their off-work hours-by avoidance, situating incidents within an analysis of the circumstances of elderly clients, setting boundaries on discussions, and occasionally, confrontation.
Asunto(s)
Diversidad Cultural , Auxiliares de Salud a Domicilio , Servicios Domésticos , Prejuicio , Mujeres Trabajadoras , Adulto , Anciano , Emigración e Inmigración , Etnicidad , Femenino , Grupos Focales , Humanos , Persona de Mediana Edad , Modelos Organizacionales , Ontario , Filipinas/etnología , Política Pública , Factores Socioeconómicos , Estereotipo , Indias Occidentales/etnología , Recursos Humanos , Lugar de TrabajoRESUMEN
The pathogenesis of Venezuelan equine encephalitis virus (VEE) was examined in the mouse model using V3000, a virus derived from a molecular clone of the Trinidad donkey strain of VEE. These results were compared in parallel experiments with avirulent mutants of VEE derived by site-directed mutagenesis of the clone. Adult mice, inoculated subcutaneously in their left rear footpad with V3000, were followed in a time course study for 6 days in which 15 organs were tested for histopathological changes, for the presence of viral antigen by immunohistochemical staining, for the presence of viral nucleic acid by in situ hybridization analysis, and for content of viable virus. Virus was detected in the footpad inoculation site, but until 12 hr postinoculation (pi), the level of virus did not suggest early viral replication. By 4 hr pi, however, replication of V3000 was evident in the draining popliteal lymph node. At this early time point, no virus could be isolated from any other organ examined. At 12 hr, a significant serum viremia was observed, and virus was detected at a low level in a number of well vascularized organs, including spleen, heart, lung, liver, kidney, and adrenal gland. By 18 hr, high virus titers were present in serum and all the lymphoid organs examined, and these tissues appeared to be the major peripheral sites of V3000 replication. Virus in serum and peripheral organs was cleared by 3-4 days pi. In a second phase of the infection, V3000 invaded the central nervous system (CNS), replicated predominantly in neurons, and persisted in the brain until death by encephalitis. Pathologic findings as well as the results of immunocytochemical and in situ hybridization examination were generally coordinate with virus titration. A site-directed mutant of V3000, V3010, contained a mutation in the gene for the E2 glycoprotein at codon 76 (Glu to Lys) which rendered it avirulent after footpad inoculation. Detection of V3010 replication in the draining lymph node was sporadic and was sometimes delayed to as long as 3 days pi. Infrequent and/or delayed virus spread to other sites also was observed. Analogous experiments were performed with other mutants which were avirulent by the footpad inoculation route: V3014, a mutant differing from V3000 at three loci (E2 Lys 209, E1 Thr 272, and E2 Asn 239), as well as single-site mutants V3032 (E2 Lys 209) and V3034 (E1 Thr 272).(ABSTRACT TRUNCATED AT 400 WORDS)