RESUMO
INTRODUCTION: Haemophilia B is an X-linked bleeding disorder caused by a coagulation factor IX gene (F9) abnormality. Numerous F9 defects have been identified to date; however, only a few with an entire F9 deletion have been reported in detail. AIM: To elucidate the cause of severe haemophilia B, we investigated the precise X chromosome abnormalities in four Japanese patients who did not show all amplifications in F9-specific PCR. METHODS: We analysed the patient's genomic DNA using Multiplex ligation-dependent probe amplification (MLPA). To assess the extent of any deletions, we further performed mapping PCRs, inverse PCRs or long-range PCRs and direct sequencing analyses of the X chromosome. RESULTS: We detected entire F9 deletions in four haemophilia B patients and identified the precise deleted regions of the X chromosome including F9. Patient 1 had a 149-kb deletion with breakpoints 90-kb upstream and 30-kb downstream from F9. Patients 2 and 3 showed 273-kb and 1.19-Mb deletions respectively. Patient 4 had two deleted regions: a 1663-bp deletion 1.34-Mb upstream from F9 and a 7.2-Mb deletion including F9. These distinct breakpoints found in four different patients suggest that the mechanism of X chromosome deletion may be different between individuals. Non-allelic homologous recombination (NAHR), microhomology-mediated break-induced replication (MMBIR) or fork stalling and template switching (FoSTeS) may occur in respective X chromosomes of the four haemophilia B patients analysed. CONCLUSIONS: We identified diverse X chromosomal rearrangements in four haemophilia B patients, which might be caused by distinct mechanisms of genomic rearrangement.
Assuntos
Cromossomos Humanos X , Fator IX/genética , Hemofilia B/genética , Adolescente , Adulto , Sequência de Bases , Criança , DNA/química , DNA/genética , DNA/metabolismo , Análise Mutacional de DNA , Rearranjo Gênico , Humanos , Japão , Masculino , Reação em Cadeia da Polimerase Multiplex , Deleção de Sequência , Adulto JovemRESUMO
Little is known about the neuropathogenicity of equine herpesvirus-1 (EHV-1) in mice. No neurological signs were observed in 6-day-old mice inoculated intracerebrally with the HH1 strain (HH1) of EHV-1. However,6-day-old mice inoculated intracerebrally with a variant derived by serial passage of HH1 in mouse brain showed severe neurological symptoms and eventually died. Histological analyses were performed on 6-day-old mice inoculated with the neuroadapted HH1 (NHH1) and the parental HH1 strain by the intracerebral, intranasal or intraperitoneal route. All routes of inoculation with NHH1 caused encephalitis, but myelitis was observed only in mice inoculated intraperitoneally. Prominent histological findings were perivascular cuffing sometimes associated with small fibrin thrombi, neuronal and glial degeneration and necrosis, and intranuclear inclusion bodies in neurons, glial cells and ependymal cells. Intracerebral and intranasal inoculation, but not intraperitoneal inoculation, with HH1 induced central nervous system (CNS) lesions that were milder than those in mice inoculated with NHH1. The distribution of viral antigen was more widespread in mice inoculated with NHH1 than with HH1. No viral antigen was detected in the CNS of mice inoculated intraperitoneally with HH1. These results indicate that increased viral multiplication and spreading in the CNS were responsible for the enhanced neurovirulence of NHH1. Although EHV-1 has been considered to be primarily endotheliotropic in horses, both NHH1 and HH1 showed tropism for the parenchymal cells of the CNS in mice, namely neurons, glial cells and ependymal cells.
Assuntos
Encéfalo/virologia , Encefalite Viral/patologia , Infecções por Herpesviridae/patologia , Herpesvirus Equídeo 1/patogenicidade , Medula Espinal/virologia , Administração Intranasal , Animais , Animais Lactentes , Encéfalo/patologia , Modelos Animais de Doenças , Suscetibilidade a Doenças , Encefalite Viral/transmissão , Infecções por Herpesviridae/transmissão , Herpesvirus Equídeo 1/fisiologia , Cavalos , Imuno-Histoquímica , Injeções Intraperitoneais , Injeções Intraventriculares , Camundongos , Camundongos Endogâmicos BALB C , Medula Espinal/patologia , Replicação ViralRESUMO
Intrauterine infection with equine herpesvirus-1 (EHV-1) has been considered to be the consequence of transplacental transmission of the virus following maternal cell-associated viraemia. In this study the state of EHV-1 gene expression in the placenta of seven naturally aborted equine fetuses was examined. Neither lesions nor viral antigens were detected in the placenta of the fetuses. The amount of infectious virus in the placentas was considerably lower than that in the fetal lungs, which showed pneumonia and typical herpesvirus inclusions. Quantitative dot blot hybridization with probes specific for immediate-early (IE), early (ICP0), and late (gD and gK) genes revealed that the placentas expressed the IE gene at a level comparable with that in the lungs; however, expression of the ICP0, gD and gK genes was significantly weaker in the placentas than in the lungs. In-situ hybridization demonstrated that both IE and gK RNAs were distributed mainly in the cytoplasm of trophoblasts. These results suggest that the low level of early and late gene transcription may be related to the limited production of viral progeny and the lack of immunoreactivity for viral antigen in trophoblasts infected with EHV-1.
Assuntos
Aborto Animal/virologia , Feto/virologia , Genes Virais/genética , Infecções por Herpesviridae/veterinária , Herpesvirus Equídeo 1/genética , Doenças dos Cavalos/virologia , Trofoblastos/virologia , Aborto Animal/patologia , Animais , DNA Viral/genética , Feminino , Feto/metabolismo , Feto/patologia , Regulação Viral da Expressão Gênica , Infecções por Herpesviridae/patologia , Infecções por Herpesviridae/virologia , Herpesvirus Equídeo 1/crescimento & desenvolvimento , Herpesvirus Equídeo 1/isolamento & purificação , Doenças dos Cavalos/patologia , Cavalos , Técnicas Imunoenzimáticas/veterinária , Hibridização In Situ/veterinária , RNA Viral/genética , Trofoblastos/metabolismoRESUMO
Equine herpesvirus 1 (EHV-1) shows endotheliotropism in the central nervous system (CNS) of infected horses. However, infection of endothelial cells has not been observed in the CNS of infected mice. To explore the basis for this difference in endotheliotropism, we compared the susceptibility of equine brain microvascular endothelial cells (EBMECs) and mouse brain microvascular endothelial cells (MBMECs) to EHV-1 infection. The kinetics of viral growth in EBMECs was typical of a fully productive infection whereas viral infection in MBMECs seemed to be nonproductive. Immunofluorescence microscopy using anti-EHV-1 polyclonal antibody demonstrated viral antigen in infected EBMECs, but not infected MBMECs. EHV-1 immediate early (IE), early (ICP0), and late (gB, gD and gK) transcripts were expressed in infected EBMECs. However, none of these genes was detected in infected MBMECs by reverse transcription-polymerase chain reaction. Electron microscopic examination at the stage of viral entry showed that viral particles were present within uncoated vesicles in the cytoplasm of EBMECs, but absent from those of MBMECs. These results suggest that viral entry is an important determinant of the susceptibility of EBMECs and MBMECs to EHV-1 infection.