RNA-seq analysis of latently-infected CD8+ T lymphocytes during bovine malignant catarrhal fever reveals unconventional TCR cell activation

Abstract

peer reviewedAlcelaphine herpesvirus 1 (AlHV-1) persists in wildebeest asymptomatically but induces malignant catarrhal fever (MCF) upon transmission to other ruminant species, including cattle. We have previously shown that MCF is a lethal disease resembling a peripheral T cell lymphoma (PTCL) and induced after ORF73-dependent latency establishment in CD8+ T lymphocytes. However, how AlHV-1 infection leads to CD8+ T lymphocyte proliferation and activation remains largely unknown. In this study, we investigated viral and cellular transcriptomes of bovine CD8+ T lymphocytes during MCF. Three groups of four 4-month-old calves were mock-infected or infected intranasally with 105 PFU of the virulent C500 wildtype (WT) strain or non-pathogenic ORF73-deficient (73null) AlHV-1 strain. At day 45 after infection (mock and 73null groups) or at time of MCF development (WT group), CD8+ T lymphocytes were isolated from peripheral blood to high purity before RNA extraction. Illumina Truseq stranded mRNA libraries were obtained and subjected to NextSeq500 sequencing. Transcriptomes were analysed for both viral and cellular transcripts. We took advantage of the non-pathogenic 73null strain to differentiate anti-viral effector/memory responses in CD8+ T cells from transcriptomics changes due to latent infection of these cells during MCF. While no viral transcripts could be detected in CD8+ T cells of mock or 73null-infected animals, expression of viral genes of interest in MCF-developing calves was detected including AlHV-1-specific semaphorin-like A3, undefined A4, bZIP protein A6, interleukin 4-like protein A9.5 and undefined glycoprotein A10. When analysing differential cellular gene expression (P < 10−4; change in expression of over fourfold), we observed upregulation of 151 (WT vs Mock) and 72 (WT vs 73null) genes, including Gzma, SerpinB9, Moxd1, Tox2, IL-10, and Cxcr3; and downregulation of 205 (WT vs Mock) and 271 (WT vs 73null) genes, including αv or β3 integrins. Intriguingly, while gene-set enrichment for TCR engagement was observed in CD8+ T cells from MCF-developing calves, supporting T cell activation, we also observed strong downregulation of gene transcripts normally involved in canonical TCR activation such as Cbl, Tec family tyrosine kinases Rlk, Tec, and Itk, and Syk. These data suggest unconventional TCR triggering and provide unprecedented mechanistic insights on how AlHV-1 could induce a PTCL-like disease. Future work should further determine the actual pathway(s) involved and which viral gene(s) drive T cell dysregulation