RESUMO
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.
Assuntos
Citomegalovirus/genética , Citomegalovirus/metabolismo , RNA Mensageiro/metabolismo , Replicação de Sequência Autossustentável/métodos , Proteínas Virais/metabolismo , Células Cultivadas/virologia , Citomegalovirus/imunologia , Infecções por Citomegalovirus/virologia , Glicoproteínas , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Proteínas de Membrana , RNA Mensageiro/genética , RNA Viral/genética , RNA Viral/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Proteínas Virais/genéticaRESUMO
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.
Assuntos
Infecções por Citomegalovirus/sangue , Citomegalovirus/genética , Regulação Viral da Expressão Gênica , Proteínas Imediatamente Precoces/genética , Transplante de Pulmão/fisiologia , Proteínas de Ligação a RNA/genética , Proteínas Virais/genética , Antígenos Virais/sangue , Antivirais/uso terapêutico , Citomegalovirus/imunologia , Citomegalovirus/isolamento & purificação , Infecções por Citomegalovirus/diagnóstico , Infecções por Citomegalovirus/tratamento farmacológico , Primers do DNA , Sondas de DNA , Quimioterapia Combinada , Foscarnet/uso terapêutico , Ganciclovir/uso terapêutico , Glicoproteínas , Rejeição de Enxerto/tratamento farmacológico , Humanos , Proteínas Imediatamente Precoces/sangue , Imunossupressores/uso terapêutico , Cinética , Transplante de Pulmão/imunologia , Proteínas de Membrana , Fosfoproteínas/genética , Complicações Pós-Operatórias/virologia , RNA Mensageiro/genética , Proteínas de Ligação a RNA/sangue , Fatores de Tempo , Transativadores/sangue , Transativadores/genética , Transcrição Gênica , Proteínas da Matriz Viral/genética , Proteínas Virais/sangueRESUMO
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.
Assuntos
Infecções por Citomegalovirus/virologia , Proteínas Imediatamente Precoces/genética , Transplante de Pulmão/efeitos adversos , RNA Mensageiro/sangue , Replicação de Sequência Autossustentável/métodos , Proteínas Virais/genética , Anticorpos Antivirais/sangue , Citomegalovirus/genética , Citomegalovirus/isolamento & purificação , Infecções por Citomegalovirus/diagnóstico , Humanos , Proteínas Imediatamente Precoces/metabolismo , Fosfoproteínas/sangue , RNA Mensageiro/genética , RNA Viral/sangue , RNA Viral/genética , Proteínas da Matriz Viral/sangue , Proteínas Virais/metabolismo , Viremia/virologiaRESUMO
While analyzing human cytomegalovirus (HCMV) gene expression in infected cells by RNA-specific nucleic acid sequence-based amplification (NASBA), positive results were observed for HCMV RNA encoded by several viral genes immediately after the addition of the virus. UV-inactivated virus also gave a positive NASBA result without establishing active infection, suggesting that RNA was associated with the inoculum. Highly purified virions devoid of cellular contamination proved to be positive for viral RNA encoding both immediate-early (UL123) and late (UL65) gene products. Virion-associated RNA might be incorporated specifically or without selection during the virion assembly. In the latter case, cellular RNA would also be present in the virion. A high-abundant cellular RNA encoded by GAPDH and even U1A RNA, which is expressed at low levels, were detected in the virion fraction, whereas cellular DNA was absent. Virion fractionation revealed that cellular RNA was absent in purified de-enveloped capsids. In conclusion, cellular and viral RNA was present between the capsid and envelope of the virion, whereas in the capsid only viral RNA could be detected. The results suggest that virion-associated viral and cellular RNA is incorporated nonspecifically during virion assembly.
