Department of Pathobiology, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL 61802, USA.
Viruses. 2024 May 15;16(5):782. doi: 10.3390/v16050782.
Marek's disease (MD), caused by (GaAHV2) or Marek's disease herpesvirus (MDV), is a devastating disease in chickens characterized by the development of lymphomas throughout the body. Vaccine strains used against MD include 3 (GaAHV3), a non-oncogenic chicken alphaherpesvirus homologous to MDV, and homologous meleagrid alphaherpesvirus 1 (MeAHV1) or turkey herpesvirus (HVT). Previous work has shown most of the MDV gC produced during in vitro passage is secreted into the media of infected cells although the predicted protein contains a transmembrane domain. We formerly identified two alternatively spliced gC mRNAs that are secreted during MDV replication in vitro, termed gC104 and gC145 based on the size of the intron removed for each (gC) transcript. Since gC is conserved within the subfamily, we hypothesized GaAHV3 (strain 301B/1) and HVT also secrete gC due to mRNA splicing. To address this, we collected media from 301B/1- and HVT-infected cell cultures and used Western blot analyses and determined that both 301B/1 and HVT produced secreted gC. Next, we extracted RNAs from 301B/1- and HVT-infected cell cultures and chicken feather follicle epithelial (FFE) skin cells. RT-PCR analyses confirmed one splicing variant for 301B/1 gC (gC104) and two variants for HVT gC (gC104 and gC145). Interestingly, the splicing between all three viruses was remarkably conserved. Further analysis of predicted and validated mRNA splicing donor, branch point (BP), and acceptor sites suggested single nucleotide polymorphisms (SNPs) within the 301B/1 transcript sequence resulted in no gC145 being produced. However, modification of the 301B/1 gC145 donor, BP, and acceptor sites to the MDV sequences did not result in gC145 mRNA splice variant, suggesting mRNA splicing is more complex than originally hypothesized. In all, our results show that mRNA splicing of avian herpesviruses is conserved and this information may be important in developing the next generation of MD vaccines or therapies to block transmission.
马立克氏病（MD）由（GaAHV2）或马立克氏病疱疹病毒（MDV）引起，是一种毁灭性疾病，可导致鸡全身发生淋巴瘤。用于预防 MD 的疫苗株包括 3（GaAHV3），这是一种与 MDV 同源的非致癌性鸡α疱疹病毒，以及同源的火鸡疱疹病毒 1（MeAHV1）或鸡新城疫病毒（HVT）。先前的研究表明，尽管预测蛋白包含一个跨膜结构域，但在体外传代过程中产生的大多数 MDV gC 都会分泌到感染细胞的培养基中。我们之前在 MDV 体外复制过程中鉴定了两种分泌的剪接 gC mRNA，根据每个（gC）转录物去除的内含子大小，分别命名为 gC104 和 gC145。由于 gC 在 亚科内保守，我们假设 GaAHV3（株 301B/1）和 HVT 也因 mRNA 剪接而分泌 gC。为了解决这个问题，我们收集了 301B/1-和 HVT 感染的细胞培养物的培养基，并使用 Western blot 分析确定 301B/1 和 HVT 都产生了分泌型 gC。接下来，我们从 301B/1-和 HVT 感染的细胞培养物和鸡羽毛滤泡上皮（FFE）皮肤细胞中提取 RNA。RT-PCR 分析证实 301B/1 gC 有一种剪接变体（gC104），HVT gC 有两种变体（gC104 和 gC145）。有趣的是，所有三种病毒的剪接都非常保守。对预测和验证的 mRNA 剪接供体、分支点（BP）和受体位点的进一步分析表明，301B/1 转录本序列中的单核苷酸多态性（SNP）导致没有产生 gC145。然而，将 301B/1 gC145 的供体、BP 和受体位点修饰为 MDV 序列并没有导致 gC145 mRNA 剪接变体，这表明 mRNA 剪接比最初假设的要复杂。总之，我们的研究结果表明，禽疱疹病毒的 mRNA 剪接是保守的，这一信息可能对开发新一代 MD 疫苗或阻断传播的治疗方法很重要。
