![]() | Only 14 pages are availabe for public view |
Abstract The present study include three experiment the first one concerning the equine herpesvirus 1 (EHV-1) while the second and the third one concerning the newly isolated herpesvirus named equine herpesvirus9 (EHV-9 ) . The first experiment on EHV-1 is concerned in detection of the sub-cellular localization of US9 ( ORF 76) through construction of recombinant virus containing GFP-ORF76 through cloning the orf76 in EGFP-C1 plasmid then homologus recombination to the GFP-ORF76 to the rpsl in Ab4p strain then regeneration of the infectious virus containing the fused GFP then detection of the time of protein expression and the localization using the cono-focal laser microscope and the result indicate that US9 is early expressed protein localized predominantly to the Trans -Golgi network (TGN) region. The seconed experiment is concerned on studying the pathogenesis of the EHV-9 in induction of the abortion in a hamster model through time points evaluation after experimental infections in early and late trimesters and following up the viral progress in both fetal (brain ,lung and liver) and maternal tissue (brain ,blood ,uterus and placenta ) through conventional PCR , real time PCR , histopathology and immunohistochemistry, also dtetecting the level of different inflammatory mediators associated with abortion using ELISA technique . The result indicated that the clinical signs of EHV-9 is greatly similar to EHV-1 concerning the respiratory signs ,nervous signs and the time of abortion is also late abortion as in EHV-1, The samples were collected at three time points following early infection and two time points following late infection , the pregnant hamsters were sampled for blood for viremia ,brain , lung ,liver ,placent ,uterus while the aborted feti sampled for brain ,lung and liver for viral DNA detection . the PCR result showed that the viremia was more prominent during the 3rd to the 6th day post infection while cannot be detected after that while the The viral DNA can be detected in the brain tissue from three days post infection and in all time points till full term of gestation. The viral DNA can be detected in the uterine tissue earlier after late trimester infection than in early trimester infection starting from six days post early trimester infection while three days post late trimester infection. The PCR on the placental tissue revealed that the viral DNA can be detected in all samples at full term of gestation and can be detected in one sample at three days post late trimester infection while the viral DNA on fetal tissue (brain, lung and liver) at early and late trimester in our couldn’t be detected except for two fetal liver samples at late trimester infected group . The real time result concerning the viral DNA in blood revealed significant increase between the viral load a at six days post early infection (early mid ) and the same time point following late infection ( late mid) , also significant increase at six days post late infection (late final ) and the same time point following early infection ( early final) , while the viral load in brain tissue revealed significant increase with the time progress in each time point than the previous points ,while in both uterine and placental tissue revealed significant increase in the viral load at late time of gestation than early points , while the viral DNA could be detected only in two fetal liver samples and the load was significantly increased than dam liver sample at the same time points .The result of two inflammatory mediators by ELISA revealed significant increase in the level of interferon gamma after infection and at time of abortion while the level of tumor necrotizing factor showed significant increase at time of abortion than the control after both early and late trimester infection . The third experiment is concerned in studying the neuropathogenesis of the equine herpesvirus 9 (EHV-9), a new neurotropic equine herpesvirus and its mutant clone (SP21) in a hamster model after intranasal inoculation using the same dose . Clinically, The EHV-9 infected group began to show the clinical signs at the third day post-inoculation, while the SP21 group showed the clinical signs at the fourth day post- inoculation , the clinical signs were more sever in the EHV-9 infected group than SP21 infected group . There was one animal dead in the EHV-9 infected group at the fifth day post infection while no mortality detected in the SP21 infected group till the end of the experiment. The DNA extracted from the different brain parts at different time points of the experiment from both EHV-9 and SP21 infected group revealed difference in the time of the anterograde transmission of both EHV-9 and SP21, Also the viremia could be detected in the EHV-9 infected group in the fourth and fifth day post infection while couldn’t be detected at the SP21 infected group during the experiment. The level of the tumor necrotizing factor –alpha detected by ELISA at days post infection revealed significant increase in the EHV-9 infected group than sp21 infected group at the fourth and fifth days post infection while no significant difference between the two infected groups in the level of interferon –gamma . Remarkably similar spatiotemporal profiles of virus replication and virus-associated histopathology in brain that were high in olfactory bulb and cerebral hemispheres but progressively decreased toward the medulla oblongata and the spinal cord. Histopathological analysis revealed that the mean group scores for the entire brain were significantly higher for hamsters inoculated with EHV-9 than for hamsters inoculated with SP21 at all-time points starting from 3dpi. This study provides a new data about the neuropathogenesis pattern of the EHV-9 in the brain and the related cytokines profile. These results suggest that the gene products of ORF19 and ORF14 play important role in the neuropathogenecity of the EHV-9 as the two point mutation detected in SP21 greatly affects its neuropathogenesis. |