Project description:Evidence supporting the role of pathogen exposure in the development of dementia has been mounting for decades. We used murine cytomegalovirus (MCMV) as our model pathogen. We repeated infected mice every 3 months modeling the re-activation/re-infection with MCMV that occurs throughout life. We used Multiome ATAC-seq and RNA-seq to examine changes to the brain of infected animals.
Project description:Comparison of gene expression profiles of human foreskin fibroblasts (HFF) infected with 3 clinical isolates of cytomegalovirus strains representing three glycoprotein B genotypes. Keywords: other
Project description:Purpose: To characterize transcriptional profiles of murine cytomegalovirus infected allografts after renal transplantation. Methods: RNA was isolated from murine allografts and native kidneys, with and without MCMV infection. Libraries were generated and paired end 150 base pair sequencing was performed on the HiSeq 4000 (Illumina) (Supplementary Methods). Each sample was aligned to the GRCm38.p4 assembly of the mouse reference from NCBI using version 2.6.0c of the RNA-Seq aligner STAR. Transcript features were identified from the GFF file provided with the assembly from NCBI and raw coverage counts were calculated using HTSeq. The raw RNA-Seq gene expression data was normalized and post-alignment statistical analyses were performed using DESeq2 and custom analysis scripts written in R. Comparisons of gene expression and associated statistical analysis were made between different conditions of interest using the normalized read counts. All fold change values are expressed as test condition/control condition, where values less than 1 are denoted as the negative of its inverse. Results: The QIAGEN Ingenuity Pathway Analysis (IPA) software was used for canonical pathway and differential gene expression analyses. IPA showed that, compared to MCMV infected native kidneys, transplantation of MCMV-infected kidneys led to significant changes in 5502 genes (adjusted p values <0.05), involved in 391 canonical pathways. The Th17 activation pathway showed 107 differentially expressed genes.Th1 pathway was one of the most highly upregulated pathways observed in the MCMV infected allografts. Conclusions: Transcripts for Th1/Th17 cell associated activation and signaling are differentially expressed in MCMV infected kidneys after allogeneic transplantation.
Project description:We characterized the role of the conserved murine cytomegalovirus (MCMV) gene M79. Using a recombinant MCMV virus carrying a tagged M79 coding sequence, we showed that M79 encoded a protein (pM79) which was expressed at late times of infection and localized to nuclear viral replication compartments. M79 transcription was largely dependent on viral DNA synthesis but was markedly stimulated by pM79, suggesting a positive feedback loop. To investigate its role, we created the recombinant virus SMin79, in which the M79 coding sequence was disrupted by an 88-nt insertion. We subsequently repaired the mutation to generate marker-rescued virus SMrev79. While SMrev79 grew efficiently in fibroblasts, SMin79 failed to produce infectious progeny but was rescued by pM79 expression in trans. During SMin79 infection, representative viral immediate early and early gene products, as well as viral DNA, accumulated efficiently. Formation of viral replication compartments also appeared normal. Pulsed field gel electrophoresis analysis indicated that the overall structure of replicating viral DNA was indistinguishable in cells infected with SMin79 compared to that with wild type virus. However, the accumulation of viral products for late gene M55 was severely compromised. Viral oligonucleotide tiled array analysis revealed that the accumulation of many late transcripts, defined by their sensitivity to viral DNA synthesis inhibitor phosphonoacetic acid, was markedly reduced by pM79 mutation. This study extends our previous work to suggest that cytomegaloviruses use a conserved mechanism to promote transcription at late stages of infection, and that pM79 regulates expression of a subset of viral DNA synthesis-dependent transcripts. This study consists of 4 unreplicated samples. RNA from Mock-infected, WT infected, WT infected in the presence of PAA, and a mutant M79 version of MCMV.
Project description:Using Next-generation sequencing of total RNA isolated from human cytomegalovirus virions we have analyzed host (human) snoRNA molecules.