Marek’s disease virus (MDV) is a strictly cell-associated herpesvirus that is deadly and oncogenic in its natural host with up to 100% mortalities in unvaccinated flocks. In addition to the economic and agriculture importance, it is a natural virus-host model for the investigation of virus-induced lymphomas. Lymphomas are the main cause of MDV-induced tremendous suffering and death in chickens. MDV harbours a linear double-stranded genome of about 180-kilobase pairs composed of two unique regions, unique long (UL) and unique short (US). Unique regions are flanked by further repeats that have a set of genes that contributes to MDV pathogenicity and oncogenicity. These genes are the major oncogene meq, the open reading frames (ORF) RLORF5a and RLORF4, and the chemokine homolog vIL-8. Moreover, these genes encode a set of splice variants that are thought to contribute to MDV pathogenesis and carcinogenesis. Yet it is unclear if all or some of these splice variants encode an expressed protein. Interestingly, these variants are spliced to the vIL-8 gene that consists of three exons and two introns. Introns are non-coding sequences and yet their significance is underappreciated or poorly understood, despite they include important junctions that mediate splicing. In this study, one or all introns of vIL-8 were removed to abrogate the splicing of vIL-8 and characterize the importance of introns for virus replication and protein secretion. Interestingly, vIL-8 introns were non-essential for virus replication, but they were indispensable for protein secretion. In addition, intron II was found to harbour a novel exon (E3`) that is spliced to express novel vIL-8 protein variants that were non-essential for virus replication or protein secretion. This study is the first to report that alphaherpesviruses express novel chemokine variants and lay the foundation for future studies to dissect the role of vIL-8 splice variants in MDV pathogenesis and tumour formation. CRISPR/Cas was discovered in bacteria and archaea as an adaptive antiviral immune mechanism. Streptococcus pyogenes have evolved CRISPR/Cas9, which is the best-studied example of the CRISPR/Cas systems until now. CRISPR/Cas9 has been recently used in various applications that revolutionized the basic molecular biology. In principle, guide RNAs recruit Cas9 to cleave the targets in a very precise manner. In this study, CRISPR/Cas9 was used to target MDV essential genes to inhibit virus replication. Interestingly, while single gRNAs significantly impaired virus replication to different degrees, multiple gRNAs completely inhibited MDV replication. Moreover, CRISPR/Cas9 was tested for the long-term elimination of MDV from infected cultures that were serially passaged. Intriguingly, the single guides showed MDV escape mutants that appeared very quickly, yet the combined gRNAs did not produce MDV that is able to evade CRISPR/Cas9. Sequencing of the escape mutants revealed specific mutations that were seen in the recognition sites of two MDV single gRNAs, which would eliminate their ability to functionally cleave the mutated MDV targets. In all, this is the first demonstration that CRISPR/Cas9 can inhibit MDV replication and evasion, especially when two or more gRNAs are used. This study provides the basis for future studies to protect chickens from this deadly oncogenic herpesvirus.