Deciphering the role of telomeric repeats encoded by Marek’s disease virus-vaccines in latency, integration and protection against disease

马立克氏病病毒（MDV）是一种高致癌性的甲疱疹病毒，可引起鸡的免疫抑制、神经系统症状和T细胞淋巴瘤。疫苗对每年保护数十亿鸡免受这种致命病毒的侵害至关重要。三种不同的活疫苗病毒，火鸡疱疹病毒（HVT），鸡瘿疱疹病毒3 （GaHV-3）。SB-1)和减毒的MDV菌株CVI988，用于预防MDV，也可作为针对其他病原体的疫苗载体；然而，人们对这些病毒的生物学特性几乎一无所知。我们的初步数据显示，这些疫苗病毒在潜伏感染细胞中整合到含有宿主端粒的宿主染色体末端。有趣的是，这些MDV疫苗病毒在其基因组的两端携带与宿主端粒相同的端粒重复序列（TMRs），这可能促进整合。因此，我们假设tmr促进了疫苗病毒与宿主端粒的整合，从而有助于疫苗潜伏期和由于长时间接触病毒而产生的疫苗保护。我们将以三个具体目标来解决这个假设。具体来说，我们将1)确定tmr在体外疫苗复制和整合中的作用，2)阐明tmr在宿主中建立疫苗潜伏期和再激活中的作用，以及3)研究疫苗病毒的整合和持久性是否有助于预防MDV。为了实现目标1，我们将生成缺乏TMR（∆TMR）的重组疫苗病毒（HVT、sdb -1和CVI988），利用我们最近建立的体外整合试验来评估这些病毒端粒序列在病毒复制和整合中的作用。此外，我们将利用我们的新型Bionano成像方法研究野生型和∆TMR疫苗病毒的整合位点。为了实现目标2，我们将用野生型和∆TMR疫苗（HVT-∆TMR已经可用）感染鸡，并随着时间的推移监测病毒的复制、传播和脱落到环境中。除此之外，我们将研究淋巴器官中存在和不存在tmr的潜伏期和再激活。在目标3中，我们将给鸡接种野生型和∆TMR疫苗（HVT-∆TMR已经可用），并用一种毒性很强的MDV毒株攻击它们。我们将评估这些疫苗在抑制MDV复制和脱落方面的功效，并确定疫苗病毒整合促进的长期持久性是否有助于疫苗对MDV的保护。总而言之，该项目将为这些重要的兽医疫苗病毒的生物学提供重要的见解，包括tmr在疫苗整合、潜伏期和疫苗保护中对这种致命病原体的作用。

Marek’s disease virus (MDV) is a highly oncogenic alphaherpesvirus that causes immunosuppression, neurological symptoms and T cell lymphomas in chickens. Vaccines are crucial to protect billions of chickens against this deadly virus every year. Three different live vaccine viruses, herpesvirus of turkey (HVT), gallid herpesvirus 3 (GaHV-3, aka. SB-1) and the attenuated MDV strain CVI988, are used to protect against MDV and also serve as vaccine vectors against other pathogens; however, hardly anything is known about the biology of these viruses. Our preliminary data revealed that theses vaccine viruses integrate into the ends of host chromosomes containing the host telomeres in latently infected cells. Intriguingly, these MDV vaccine viruses harbor telomeric repeat arrays (TMRs) identical to the host telomeres at both ends of their genomes, which could facilitate integration. We therefore hypothesize that the TMRs facilitate integration of the vaccine viruses into host telomeres, and thereby contribute to vaccine latency and vaccine protection due to prolonged exposure with the virus. We will address this hypothesis with three specific aims. Specifically, we will 1) determine the role of the TMRs in vaccine replication and integration in vitro, 2) elucidate the role of the TMRs in the establishment of vaccine latency and reactivation in the host and 3) investigate if integration and persistence of the vaccine viruses contribute to the protection against MDV. To achieve aim 1, we will generate recombinant vaccine viruses (HVT, SB-1 and CVI988) lacking TMRs (∆TMR) to assess the role of these viral telomere sequences in virus replication and integration using our recently established in vitro integration assay. Further, we will investigate the integration sites of wild type and ∆TMR vaccine viruses using our novel Bionano imaging approach. To address aim 2, we will infect chickens with wild type and ∆TMR vaccines (HVT-∆TMR already available) and monitor virus replication, dissemination and shedding into the environment over time. Beyond that, we will investigate latency and reactivation in the presence and absence of the TMRs in the lymphoid organs. In aim 3, we will vaccinate chickens with wild type and ∆TMR vaccines (HVT-∆TMR already available) and challenge them with a very virulent MDV strain. We will assess the efficacy of these vaccines in suppressing MDV replication and shedding, and determine if prolonged persistence facilitated by vaccine virus integration contributes to vaccine-protection against MDV. Taken together, this project will provide crucial insights into the biology of these important veterinary vaccine viruses including the roles of the TMRs in vaccine integration, latency and vaccine protection against this deadly pathogen.