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Duck enteritis virus (DEV) is an important herpesvirus pathogen associated with acute, highly contagious lethal disease in waterfowls. Using a deep sequencing approach on RNA from infected chicken embryo fibroblast cultures, we identified several novel DEV-encoded micro (mi)RNAs. Unlike most mardivirus-encoded miRNAs, DEV-encoded miRNAs mapped mostly to the unique long region of the genome. The precursors of DEV miR-D18 and miR-D19 overlapped with each other, suggesting similarities to miRNA-offset RNAs, although only the DEV-miR-D18-3p was functional in reporter assays. Identification of these novel miRNAs will add to the growing list of virus-encoded miRNAs enabling the exploration of their roles in pathogenesis.

The identification of specific genetic changes associated with differences in the pathogenicity of Marek's disease virus strains (GaHV-2) has been a formidable task due to the large number of mutations in mixed-genotype populations within DNA preparations. Very virulent UK isolate C12/130 induces extensive lymphoid atrophy, neurological manifestations and early mortality in young birds. We have recently reported the construction of several independent full-length bacterial artificial chromosome (BAC) clones of C12/130 capable of generating fully infectious viruses with significant differences in their pathogenicity profiles. Two of these clones (vC12/130-10 and vC12/130-15), which showed differences in virulence relative to each other and to the parental strain, had similar replication kinetics both in vitro and in vivo in spite of the fact that vC12/130-15 was attenuated. To investigate the possible reasons for this, the nucleotide sequences of both clones were determined. Sequence analysis of the two genomes identified mutations within eight genes. A single 494 bp insertion was identified within the genome of the virulent vC12/130-10 clone. Seven non-synonymous substitutions distinguished virulent vC12/130-10 from that of attenuated vC12/130-15. By sequencing regions of parental DNA that differed between the two BAC clones, we confirmed that C12/130 does contain these mutations in varying proportions. Since the individual reconstituted BAC clones were functionally attenuated in vivo and derived from a single DNA source of phenotypically very virulent C12/130, this suggests that the C12/130 virus population exists as a collection of mixed genotypes.

The complete DNA sequence of the Marek's disease virus serotype 1 vaccine strain CVI988 was determined and consists of 178 311 bp with an overall gene organization identical to that of the oncogenic strains. In examining open reading frames (ORFs), nine differ between vaccine and oncogenic strains. A 177 bp insertion was identified in the overlapping genes encoding the Meq, RLORF6 and 23 kDa proteins of CVI988. Three ORFs are predicted to encode truncated proteins. One, designated 49.1, overlaps the gene encoding the large tegument protein UL36 and encodes a severely truncated protein of 34 aa. The others, ORF5.5/ORF75.91 and ORF3.0/78.0, located in the repeat regions (diploid), encode a previously unidentified ORF of 52 aa and a truncated version of the virus-encoded chemokine (vIL-8), respectively. Subtle genetic changes were identified in the two ORFs encoding tegument proteins UL36 and UL49. Only one diploid ORF (ORF6.2/ORF75.6) present in the genomes of the three virulent strains is absent in the CVI988-BAC genome. Seventy non-synonymous amino acid substitutions were identified that could differentiate CVI988-BAC from all three oncogenic strains collectively. Estimates of the non-synonymous to synonymous substitution ratio (omega) indicate that CVI988 ORFs are generally under purifying selection (omega<1), whereas UL39, UL49, UL50, RLORF6 and RLORF7 (Meq) appear to evolve under relaxed selective constraints. No CVI988 ORF was found to be under positive evolutionary selection (omega>>1).