generating-recombinant-avian-herpesvirus-vectors-with-crisprcas9-gene

Generating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian ...

The Pirbright Institute & UK-China Centre of Excellence for Research on Avian Diseases

Several studies have shown that microRNA-targeting is an effective strategy for the selective control of tissue-tropism and pathogenesis of both DNA and RNA viruses. However, the exploitation of microRNA-targeting for the inhibition of transformation by oncogenic viruses has not been studied. The v- oncoprotein encoded by reticuloendotheliosis virus T strain (Rev-T) is a member of the /NF-&kappa;B family of transcription factors capable of transforming primary chicken spleen and bone marrow cells. Here, by engineering the target sequence of endogenous microRNA miR-142 downstream of the v- gene in a Replication-Competent ALV (avian leukosis virus) long terminal repeat (LTR) with a splice acceptor (RCAS) vector and using a v--induced transformation model of chicken embryonic splenocyte cultures, we show that hematopoietic-specific miR-142 can inhibit the v--induced transformation, and that this inhibition effect is due to the silencing of v- expression. The data supports the idea that microRNA-targeting can be used to inhibit viral oncogene-induced oncogenesis.

Inhibition of v--Induced Oncogenesis through microRNA Targeting.

Superinfection of Marek's disease virus (MDV) and avian leukosis virus subgroup J (ALV-J) causes lethal neoplasia and death in chickens. However, whether there is synergism between the two viruses in viral replication and pathogenicity has remained elusive. In this study, we found that the superinfection of MDV and ALV-J increased the viral replication of the two viruses in RNA and protein level, and synergistically promoted the expression of IL-10, IL-6, and TGF-β in chicken embryo fibroblasts (CEF). Moreover, MDV and ALV-J protein expression in dual-infected cells detected by confocal laser scanning microscope appeared earlier in the cytoplasm and the nucleus, and caused more severe cytopathy than single infection, suggesting that synergistically increased MDV and ALV-J viral-protein biosynthesis is responsible for the severe cytopathy. In vivo, compared to the single virus infected chickens, the mortality and tumor formation rates increased significantly in MDV and ALV-J dual-infected chickens. Viral loads of MDV and ALV-J in tissues of dual-infected chickens were significantly higher than those of single-infected chickens. Histopathology observation showed that more severe inflammation and tumor cells metastases were present in dual-infected chickens. In the present study, we concluded that synergistic viral replication of MDV and ALV-J is responsible for the enhanced pathogenicity in superinfection of chickens.

Synergistic Viral Replication of Marek's Disease Virus and Avian Leukosis Virus Subgroup J is Responsible for the Enhanced Pathogenicity in the Superinfection of Chickens.

Marek's disease virus (MDV) is a member of alphaherpesviruses associated with Marek's disease, a highly contagious neoplastic disease in chickens. Complete sequencing of the viral genome and recombineering techniques using infectious bacterial artificial chromosome (BAC) clones of Marek's disease virus genome have identified major genes that are associated with pathogenicity. Recent advances in CRISPR/Cas9-based gene editing have given opportunities for precise editing of the viral genome for identifying pathogenic determinants. Here we describe the application of CRISPR/Cas9 gene editing approaches to delete the  and  genes from the CVI988 vaccine strain of MDV. This powerful technology will speed up the MDV gene function studies significantly, leading to a better understanding of the molecular mechanisms of MDV pathogenesis.

Yongxiu Yao

The Pirbright Institute & UK-China Centre of Excellence for Research on Avian Diseases

Generating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing