RESUMEN
Delayed elimination of human cytomegalovirus (HCMV)-infected cells by the host immune system may contribute to viral dissemination and pathogenesis of HCMV infection. The mRNA expression dynamics of HCMV-encoded immune evasion genes US3, US6, and US11 expressed after active HCMV infection were analyzed in blood samples of lung transplant recipients by means of quantitative nucleic acid sequence-based amplification. The results were compared with the expression dynamics of IE1 mRNA and pp67 late mRNA, levels of pp65 antigenemia, and antiviral treatment. During acute infection, high levels of US3 and US6 RNA were detected before antigenemia, which were detected simultaneously with IE1 RNA. US11 RNA was detected simultaneously with antigenemia but before late pp67 RNA. These data suggest an active role of viral immune evasion during HCMV infection in vivo. Interestingly, immune evasion RNA remained detectable after clinical recovery, often independently of IE1 RNA expression, indicating persistent viral activity, which may have implications for long-term control of HCMV.
Asunto(s)
Infecciones por Citomegalovirus/sangre , Citomegalovirus/genética , Regulación Viral de la Expresión Génica , Proteínas Inmediatas-Precoces/genética , Trasplante de Pulmón/fisiología , Proteínas de Unión al ARN/genética , Proteínas Virales/genética , Antígenos Virales/sangre , Antivirales/uso terapéutico , Citomegalovirus/inmunología , Citomegalovirus/aislamiento & purificación , Infecciones por Citomegalovirus/diagnóstico , Infecciones por Citomegalovirus/tratamiento farmacológico , Cartilla de ADN , Sondas de ADN , Quimioterapia Combinada , Foscarnet/uso terapéutico , Ganciclovir/uso terapéutico , Glicoproteínas , Rechazo de Injerto/tratamiento farmacológico , Humanos , Proteínas Inmediatas-Precoces/sangre , Inmunosupresores/uso terapéutico , Cinética , Trasplante de Pulmón/inmunología , Proteínas de la Membrana , Fosfoproteínas/genética , Complicaciones Posoperatorias/virología , ARN Mensajero/genética , Proteínas de Unión al ARN/sangre , Factores de Tiempo , Transactivadores/sangre , Transactivadores/genética , Transcripción Genética , Proteínas de la Matriz Viral/genética , Proteínas Virales/sangreRESUMEN
Different cell types were infected with human cytomegalovirus (HCMV) and RNA expression dynamics were analyzed by quantitative NASBA assays for IE1 (UL123), pp67 (UL65) and the immune evasion genes (US3, US6 and US11). The quantitative NASBA assays gave reproducible quantification in the range of 10(3)-10(6) RNA copies for IE1 and pp67 RNA, from 3x10(3) to 1x10(6) RNA copies for US6 and US11 RNA, and from 1x10(4) to 1x10(6) RNA copies for US3 RNA. SMC, HAEC and HUVEC cells infected with an, in endothelial cells, propagated HCMV strain (VHL/E) showed similar RNA expression dynamics for the analyzed genes. Expression of all genes studied was observed within the first 4 h post-infection. The first gene for which expression could be detected was IE1, followed by US3, US11, pp67 and US6. Fibroblasts infected with HCMV strain AD169 showed a different RNA expression pattern for US3. Translation of the mRNA studied was demonstrated by detection of the proteins 48 h post-infection by immunofluorescence.
Asunto(s)
Citomegalovirus/genética , Citomegalovirus/metabolismo , ARN Mensajero/metabolismo , Replicación de Secuencia Autosostenida/métodos , Proteínas Virales/metabolismo , Células Cultivadas/virología , Citomegalovirus/inmunología , Infecciones por Citomegalovirus/virología , Glicoproteínas , Humanos , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Proteínas de la Membrana , ARN Mensajero/genética , ARN Viral/genética , ARN Viral/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Proteínas Virales/genéticaRESUMEN
The dynamics of active human cytomegalovirus (HCMV) infection was monitored by competitive nucleic acid sequence-based amplification (NASBA) assays for quantification of IE1 (UL123) and pp67 (UL65) mRNA expression levels in the blood of patients after lung transplantation. RNA was isolated from 339 samples of 13 lung transplant recipients and analyzed by the quantitative IE1 and pp67 NASBA in parallel with pp65 antigenemia and serology. Rapid increases in IE1 RNA exceeding 10(4) copies per 100 microl of blood were associated with active infection, whereas lower levels were suggestive for abortive, subclinical viral activity. Any positive value for pp67 RNA was indicative for active infection, and quantification of pp67 mRNA did not give additional diagnostic information. The onset of IE1-positive NASBA preceded pp67 NASBA and was earlier than the pp65 antigenemia assay, confirming previous studies with qualitative NASBA. Effective antiviral treatment was reflected by a rapid disappearance of pp67 mRNA, whereas IE1 mRNA remained detectable for longer periods. Quantification of IE1 might be relevant to monitor progression of HCMV infection but should be validated in prospective studies.