Herpesvirus of turkeys (HVT), used originally as a vaccine against Marek’s disease (MD), has recently been shown to be a highly effective viral vector for generation of recombinant vaccines that deliver protective antigens of other avian pathogens. Until the recent launch of commercial HVT-vectored dual insert vaccines, most of the HVT-vectored vaccines in the market carry a single foreign gene and are usually developed with slow and less efficient conventional recombination methods. There is immense value in developing multivalent HVT-vectored vaccines capable of inducing simultaneous protection against multiple avian pathogens, particularly to overcome the interference between individual recombinant HVT vaccines. Here we demonstrate the use of a previously developed CRISPR/Cas9 gene editing protocol for the insertion of ILTV gD-gI and the H9N2 AIV hemagglutinin expression cassettes into the distinct locations of the recombinant HVT-IBDV VP2 viral genome, to generate the triple insert HVT-VP2-gDgI-HA recombinant vaccine. The insertion, protein expression, and stability of each insert were then evaluated by PCR, immunostaining and Western blot analyses. The successful generation of the first triple insert recombinant HVT vaccine with the potential for the simultaneous protection against three major avian viral diseases in addition to MD is a major innovation in vaccination-based control of major poultry diseases.

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 Meq and pp38 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.

Marek’s disease virus (MDV), a lymphotropic α-herpesvirus associated with T-cell lymphomas in chickens, is an excellent model for herpesvirus biology and virus-induced oncogenesis. Marek’s disease (MD) is also one of the cancers against which a vaccine was first used. In the lymphomas and lymphoblastoid cell lines (LCLs) derived from them, MDV establishes latent infection with limited gene expression. Although LCLs are valuable for interrogating viral and host gene functions, molecular determinants associated with the maintenance of MDV latency and lytic switch remain largely unknown, mainly due to the lack of tools for in situ manipulation of the genomes in these cell lines. Here we describe the first application of CRISPR/Cas9 editing approach for precise editing of the viral gene phosphoprotein 38 (pp38), a biomarker for latent/lytic switch in MDV-transformed LCLs MDCC-MSB-1 (Marek’s disease cell line MSB-1) and MDCC-HP8. Contradictory to the previous reports suggesting that pp38 is involved in the maintenance of transformation of LCL MSB-1 cells, we show that pp38-deleted cells proliferated at a significant higher rate, suggesting that pp38 is dispensable for the transformed state of these cell lines. Application of CRISPR/Cas9-based gene editing of MDV-transformed cell lines in situ opens up further opportunities towards a better understanding of MDV pathogenesis and virus-host interactions.

CRISPR-Cas technology, discovered originally as a bacterial defense system, has been extensively repurposed as a powerful tool for genome editing for multiple applications in biology. In the field of virology, CRISPR-Cas9 technology has been widely applied on genetic recombination and engineering of genomes of various viruses to ask some fundamental questions about virus–host interactions. Its high efficiency, specificity, versatility, and low cost have also provided great inspiration and hope in the field of vaccinology to solve a series of bottleneck problems in the development of recombinant viral vaccines. This review highlights the applications of CRISPR editing in the technological advances compared to the traditional approaches used for the construction of recombinant viral vaccines and vectors, the main factors affecting their application, and the challenges that need to be overcome for further streamlining their effective usage in the prevention and control of diseases. Factors affecting efficiency, target specificity, and fidelity of CRISPR-Cas editing in the context of viral genome editing and development of recombinant vaccines are also discussed.

African swine fever virus (ASFV) causes a lethal swine hemorrhagic disease and is currently responsible for widespread damage to the pig industry. The pathogenesis of ASFV infection and its interaction with host responses remain poorly understood. In this study, we profiled the temporal viral and host transcriptomes in porcine alveolar macrophages (PAMs) with virulent and attenuated ASFV strains. We identified profound differences in the virus expression programs between SY18 and HuB20, which shed light on the pathogenic functions of several ASFV genes. Through integrated computational analysis and experimental validation, we demonstrated that compared to the virulent SY18 strain, the attenuated HuB20 quickly activates expression of receptors, sensors, regulators, as well as downstream effectors, including cGAS, STAT1/2, IRF9, MX1/2, suggesting rapid induction of a strong antiviral immune response in HuB20. Surprisingly, in addition to the pivotal DNA sensing mechanism mediated by cGAS-STING pathway, infection of the DNA virus ASFV activates genes associated with RNA virus response, with stronger induction by HuB20 infection. Taken together, this study reveals novel insights into the host-virus interaction dynamics, and provides reference for future mechanistic studies of ASFV pathogenicity.