Expression of Ovine Herpesvirus -2 Encoded MicroRNAs in an Immortalised Bovine – Cell Line

Ovine herpesvirus-2 (OvHV-2) infects most sheep, where it establishes an asymptomatic, latent infection. Infection of susceptible hosts e.g. cattle and deer results in malignant catarrhal fever, a fatal lymphoproliferative disease characterised by uncontrolled lymphocyte proliferation and non MHC restricted cytotoxicity. The same cell populations are infected in both cattle and sheep but only in cattle does virus infection cause dysregulation of cell function leading to disease. The mechanism by which OvHV-2 induces this uncontrolled proliferation is unknown. A number of herpesviruses have been shown to encode microRNAs (miRNAs) that have roles in control of both viral and cellular gene expression. We hypothesised that OvHV-2 encodes miRNAs and that these play a role in pathogenesis. Analysis of massively parallel sequencing data from an OvHV-2 persistently-infected bovine lymphoid cell line (BJ1035) identified forty-five possible virus-encoded miRNAs. We previously confirmed the expression of eight OvHV-2 miRNAs by northern hybridization. In this study we used RT-PCR to confirm the expression of an additional twenty-seven OvHV-2-encoded miRNAs. All thirty-five OvHV-2 miRNAs are expressed from the same virus genome strand and the majority (30) are encoded in an approximately 9 kb region that contains no predicted virus open reading frames. Future identification of the cellular and virus targets of these miRNAs will inform our understanding of MCF pathogenesis.绵羊疱疹病毒2型（Ovine herpesvirus-2, OvHV-2）可感染绝大多数绵羊，并在宿主体内建立无症状潜伏感染。易感宿主（如牛与鹿）感染该病毒后，会引发恶性卡他热（malignant catarrhal fever, MCF）——一种以淋巴细胞失控增殖、非主要组织相容性复合体（MHC）限制性细胞毒性为特征的致死性淋巴增生性疾病。牛与绵羊的感染细胞群完全一致，但仅在牛体内，病毒感染会引发细胞功能失调并最终导致疾病。目前绵羊疱疹病毒2型诱导这种细胞失控增殖的具体机制尚不明确。已有研究证实多种疱疹病毒可编码微小RNA（microRNAs, miRNAs），这类RNA在调控病毒与宿主细胞的基因表达中发挥重要作用。我们提出假说：绵羊疱疹病毒2型可编码微小RNA，且这类RNA在恶性卡他热的发病机制中具有关键功能。通过对持续感染绵羊疱疹病毒2型的牛淋巴样细胞系（BJ1035）的大规模平行测序数据进行分析，共鉴定出45种潜在的病毒编码微小RNA。此前我们已通过Northern印迹杂交（Northern hybridization）证实了8种绵羊疱疹病毒2型编码微小RNA的表达。本研究中，我们借助逆转录聚合酶链式反应（RT-PCR）进一步证实了另外27种绵羊疱疹病毒2型编码微小RNA的表达。所有35种绵羊疱疹病毒2型编码的微小RNA均由病毒基因组的同一条链转录而来，其中大部分（30种）编码于一段长约9kb的区域内，该区域未预测到病毒开放阅读框（open reading frames, ORFs）。后续对这些微小RNA的宿主与病毒靶标进行鉴定，将有助于我们深入解析恶性卡他热的发病机制。