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Marek's disease virus (MDV) causes a deadly lymphoproliferative disease in chickens, resulting in huge economic losses in the poultry industry. It has been suggested that MDV suppresses the induction of type I interferons and thus escapes immune control. Cholesterol 25-hydroxylase (CH25H), a gene that encodes an enzyme that catalyses cholesterol to 25-hydroxycholesterol (25-HC), is an interferon-stimulating gene (ISG) known to exert antiviral activities. Other oxysterols, such as 27-hydroxycholesterols (27-HC), have also been shown to exert antiviral activities, and 27-HC is synthesised by the catalysis of cholesterol via the cytochrome P450 enzyme oxidase sterol 27-hydroxylase A1 (CYP27A1). At 24 h post infection (hpi), MDV stimulated a type I interferon (IFN-α) response, which was significantly reduced at 48 and 72 hpi, as detected using the luciferase assay for chicken type I IFNs. Then, using RT-PCR, we demonstrated that chicken type I IFN (IFN-α) upregulates chicken CH25H and CYP27A1 genes in chicken embryo fibroblast (CEF) cells. In parallel, our results demonstrate a moderate and transient upregulation of CH25H at 48 hpi and CYP27A1 at 72hpi in MDV-infected CEF cells. A significant reduction in MDV titer and plaque sizes was observed in CEFs treated with 25-HC or 27-HC in vitro, as demonstrated using a standard plaque assay for MDV. Taken together, our results suggest that 25-HC and 27-HC may be useful antiviral agents to control MDV replication and spread.

Infectious bursal disease virus (IBDV) vaccines do not induce sterilizing immunity, and vaccinated birds can become infected with field strains. Vaccine-induced immune selection pressure drives the evolution of antigenic drift variants that accumulate amino acid changes in the hypervariable region (HVR) of the VP2 capsid, which may lead to vaccine failures. However, there is a lack of information regarding how quickly mutations arise, and the relative contribution different residues make to immune escape. To model IBDV antigenic drift in vitro, we serially passaged a classical field strain belonging to genogroup A1 (F52/70) ten times, in triplicate, in the immortalized chicken B cell line, DT40, in the presence of sub-neutralizing concentrations of sera from birds inoculated with IBDV vaccine strain 2512, to generate escape mutants. This assay simulated a situation where classical strains may infect birds that have suboptimal vaccine-induced antibody responses. We then sequenced the HVR of the VP2 capsid at passage (P) 5 and 10 and compared the sequences to the parental virus (P0), and to the virus passaged in the presence of negative control chicken serum that lacked IBDV antibodies. Two escape mutants at P10 had the same mutations, D279Y and G281R, and a third had mutations S251I and D279N. Furthermore, at P5, the D279Y mutation was detectable, but the G281R mutation was not, indicating the mutations arose with different kinetics.

In order to better understand differences in the outcome of infectious bursal disease virus (IBDV) infection, we inoculated a very virulent (vv) strain into White Leghorn chickens of inbred line W that was previously reported to experience over 24% flock mortality, and three inbred lines (15I, C.B4 and 0) that were previously reported to display no mortality. Within each experimental group, some individuals experienced more severe disease than others but line 15I birds experienced milder disease based on average clinical scores, percentage of birds with gross pathology, average bursal lesion scores and average peak bursal virus titre. RNA-Seq analysis revealed that more severe disease in line W was associated with significant up-regulation of pathways involved in inflammation, cytoskeletal regulation by Rho GTPases, nicotinic acetylcholine receptor signaling, and Wnt signaling in the bursa compared to line 15I. Primary bursal cell populations isolated from uninfected line W birds contained a significantly greater percentage of KUL01+ macrophages than cells isolated from line 15I birds ( p < 0.01) and, when stimulated ex vivo with LPS, showed more rapid up-regulation of pro-inflammatory gene expression than those from line 15I birds. We hypothesize that a more rapid induction of pro-inflammatory cytokine responses in bursal cells following IBDV infection leads to more severe disease in line W birds than in line 15I.

The virus-encoded microRNAs (miRNAs) have been demonstrated to have important regulatory roles in herpesvirus biology, including virus replication, latency, pathogenesis and/or tumorigenesis. As an emerging efficient tool for gene editing, the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system has been successfully applied in manipulating the genomes of large DNA viruses. Herein, utilizing the CRISPR/Cas9 system with a double-guide RNAs transfection/virus infection strategy, we have established a new platform for mutagenesis of viral miRNAs encoded by the Marek's disease virus serotype 1 (MDV-1), an oncogenic alphaherpesvirus that can induce rapid-onset T-cell lymphomas in chickens. A series of miRNA-knocked out (miR-KO) mutants with deletions of the Meq- or the mid-clustered miRNAs, namely RB-1B∆Meq-miRs, RB-1B∆M9-M2, RB-1B∆M4, RB-1B∆M9 and RB-1B∆M11, were generated from vvMDV strain RB-1B virus. Interestingly, mutagenesis of the targeted miRNAs showed changes in the in vitro virus growth kinetics, which is consistent with that of the in vivo proliferation curves of our previously reported GX0101 mutants produced by the bacterial artificial chromosome (BAC) clone and Rec E/T homologous recombination techniques. Our data demonstrate that the CRISPR/Cas9-based gene editing is a simple, efficient and relatively nondisruptive approach for manipulating the small non-coding genes from the genome of herpesvirus and will undoubtedly contribute significantly to the future progress in herpesvirus biology.

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.