Stephen J. Spatz

Attenuation of Gallid herpesvirus-2 (GaHV-2), the causative agent of Marek’s disease, can occur through serial passage of a virulent field isolate in avian embryo fibroblasts. In order to gain a better understanding of the genes involved in attenuation and associate observed changes in phenotype with specific genetic variations, the genomic DNA sequence of a single GaHV-2 virulent strain (648A) was determined at defined passage intervals. Biological characterization of these “interval-isolates” in chickens previously indicated that the ability to induce transient paralysis was lost by passages 40 and the ability to induce persistent neurological disease was lost after passage 80, coincident with the loss of neoplastic lesion formation. Deep sequencing of the interval-isolates allowed for a detailed cataloguing of the mutations that exist within a single passage population and the frequency with which a given mutation occurs across passages. Gross genetic alterations were identified in both novel and well-characterized genes and cis-acting regions involved in replication and cleavage/packaging. Deletions in genes encoding the virulence factors vLipase, vIL8, and RLORF4, as well as a deletion in the promoter of ICP4, appeared between passages 61 and 101. Three mutations in the virus-encoded telomerase which predominated in late passages were also identified. Overall, the frequency of mutations fluctuated greatly during serial passage and few genetic changes were absolute. This indicates that serial passage of GaHV-2 results in the generation of a collection of genomes with limited sequence heterogeneity.

Marek’s disease virus (MDV) is a cell-associated alphaherpesvirus that induces rapid-onset T-cell lymphomas in poultry. MDV isolates vary greatly in pathogenicity. While some of the strains such as CVI988 are non-pathogenic and are used as vaccines, others such as RB-1B are highly oncogenic. Molecular determinants associated with differences in pathogenicity are not completely understood. Comparison of the genome sequences of phenotypically different strains could help to identify molecular determinants of pathogenicity. We have previously reported the construction of bacterial artificial chromosome (BAC) clones of RB-1B from which fully infectious viruses could be reconstituted upon DNA transfection into chicken cells. MDV reconstituted from one of these clones (pRB-1B-5) showed similar in vitro and in vivo replication kinetics and oncogenicity as the parental virus. However, unlike the parental RB-1B virus, the BAC-derived virus showed inability to spread between birds. In order to identify the unique determinants for oncogenicity and the ‘‘non-spreading phenotype’’ of MDV derived from this clone, we determined the full-length sequence of pRB-1B-5. Comparative sequence analysis with the published sequences of strains such as Md5, Md11, and CVI988 identified frameshift mutations in RLORF1, protein kinase (UL13), and glycoproteins C (UL44) and D (US6). Comparison of the sequences of these genes with the parental virus indicated that the RLORF1, UL44, and US6 mutations were also present in the parental RB-1B stock of the virus. However with regard to UL13 mutation, the parental RB-1B stock appeared to be a mixture of wild type and mutant viruses, indicating that the BAC cloning has selected a mutant clone. Although further studies are needed to evaluate the role of these genes in the horizontal-spreading defective phenotype, our data clearly indicate that mutations in these genes do not affect the oncogenicity of MDV.

Comparative genomic studies of attenuated and virulent strains of Gallid herpesvirus 2 (GaHV-2) have identified 6 regions of sequence variability. These regions include the open reading frames (ORFs) encoding UL36 and UL49 and regions devoid of large ORFs (132-bp repeats, a-like sequences and the junctions flanking the unique short region). Our data indicate that the carboxyl terminus of UL36 contains regions of heterogeneity that are unique to CVI988-derived attenuated strains. A deletion of the TKSERT domain and a glycine²⁴⁵ polymorphism in the UL49 proteins were also identified in these derivatives. Phylogenetic analyses of both UL36 and UL49 sequences indicate that CVI988-derived strains partition differently from other attenuated strains (RM-1 and R2/23), indicating that additional mutations contribute to attenuation. In very virulent and very virulent plus strains a single nucleotide polymorphism (SNP) was identified within the 132-bp tandem repeats. Within the junctions flanking the unique short region, these strains also contain deletions in sequences that are predicted to bind the transcription factor NF kappaB. In some attenuated strains, deletions were also identified in the latency-associated transcript (LAT) promoters and adjacent regions encoding microRNAs. These results indicate that virulence is likely multi-factorial with contributions from both multiple genes and cis-acting sites.

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 (ω) indicate that CVI988 ORFs are generally under purifying selection (ωω≫1).

The nucleotide sequences of the terminal repeat long (TR_L) and internal repeat long regions (IR_L) in the genomes of 13 strains of Marek’s disease virus type 1 (MDV-1) were determined and represent the largest collection of sequencing data from a contiguous region (12.8 kb) in the serotype 1 genomes. The collection of strains used in this study has been well characterized with respect to their virulence and contains members of each pathotype (4 attenuated, 1 mildly virulent, 3 virulent, 2 very virulent and 3 very virulent plus). It has previously been reported that two loci (meq and RLORF4) in the RL regions are likely to encode virulence factors based on comparative genomic studies involving vaccine and virulent strains. Additional studies using knockout mutants have provided stronger evidence that indeed RLORF4 and meq or the overlapping genes 23 kD and RLORF6 are involved in virulence. In this report, we provide evidence that additional open reading frames (ORFs) in the RL regions differ significantly between the extremes of the pathotypes (attenuated vs. nonattenuated). A deletion of 10 base pairs has been identified in RLORF12 from two attenuated strains CVI988 BP-5, p48 and RM-1, p40; and the lower virulence strain JM/102W. A deletion of 40 bp was also identified in RLORF4 of the attenuated strain R2/23, passage 106. A 177 bp insertion within the meq loci has been identified in most of the attenuated strains examined. Interestingly, R2/23 did not contain this insertion but instead truncated proteins are predicted for the three overlapping ORFs (meq, 23 kD and RLORF6) due to a frameshift mutation. Single nucleotide polymorphisms (SNPs), which loosely partition between attenuated and nonattenuated strains, have been identified in the ORFs encoding RLORF12, RLORF8, meq, 23 kD, RLORF6, RLORF4, RLORF3 and ICP0 and three previously unidentified short ORFs: MHLS, MLHG and MPSG. Although no single nucleotide polymorphism in the RL regions could predict virulence, their overall contribution to virulence can now be examined in defined mutants containing additional insertions or deletions in ORFs, suspected of encoding virulence factors, identified by this research.