Phenotypic Characterization of Recombinant Marek's Disease Virus in Live Birds Validates Polymorphisms Associated with Virulence

Abstract

Marek's disease (MD) is a highly infectious lymphoproliferative disease in chickens with a significant economic impact. Mardivirus gallidalpha 2, also known as Marek's disease virus (MDV), is the causative pathogen and has been categorized based on its virulence rank into four pathotypes: mild (m), virulent (v), very virulent (vv), and very virulent plus (vv+). A prior comparative genomics study suggested that several single-nucleotide polymorphisms (SNPs) and genes in the MDV genome are associated with virulence, including nonsynonymous (ns) SNPs in eight open reading frames (ORF): UL22, UL36, UL37, UL41, UL43, R-LORF8, R-LORF7, and ICP4. To validate the contribution of these nsSNPs to virulence, the vv+MDV strain 686 genome was modified by replacing nucleotides with those observed in the vMDV strains. Pathogenicity studies indicated that these substitutions reduced the MD incidence and increased the survival of challenged birds. Furthermore, using the best-fit pathotyping method to rank the virulence, the modified vv+MDV 686 viruses resulted in a pathotype similar to the vvMDV Md5 strain. Thus, these results support our hypothesis that SNPs in one or more of these ORFs are associated with virulence but, as a group, are not sufficient to result in a vMDV pathotype, suggesting that there are additional variants in the MDV genome associated with virulence, which is not surprising given this complex phenotype and our previous finding of additional variants and SNPs associated with virulence.

Keywords: Marek’s disease; single-nucleotide polymorphisms; viral genome; virulence.

Figure 1

The EcoRV digestion of recombinant MDV 686-BAC clones. (A) Agarose gel electrophoresis image of MDV 686-BAC clones. (B) The expected EcoRV digestion pattern of MDV 686-BAC clones. (Lane 1) 686-BAC, (Lane 2) 686-BAC with TRS-TRL deletion (686∆TRS-TRL), (Lanes 3–4) two viruses independent of 686-BAC with nine single-nucleotide modifications (686 mut1, 686 mut2), (M) 1 kb Extend DNA ladder (NEB). The specific changes in fragmentations are on the right (*).

Figure 2

Survival rates of ADOL 15I₅ × 7₁ chickens infected with v686-BAC, v686∆TRS-TRL, v686 mut1, or v686 mut2. The survival rate was analyzed with the Log-rank (Mantel–Cox) test using Prism 10.0.1. (A) Survival percent of clinical trial 1 with maternal-antibody-negative birds; (B) survival percent of clinical trial 2 with maternal-antibody-positive birds. Statistical differences (p-value < 0.05) between v686∆TRS-TRL, v686 mut1, and v686 mut2 are indicated (n.s. = not significant, * p-value < 0.05, ** p-value < 0.01).

Figure 3

Survival curve of JM/102W, Md5, 648A, v686∆TRS-TRL, v686 mut1, or v686 mut2 challenged group in non-vaccinated birds. Survival curve of MDV JM/102W, Md5, 648A, v686∆TRS-TRL, v686 mut1, or v686 mut2 challenged group in HVT-vaccinated birds is in Figure S6 and survival curve of MDV JM/102W, Md5, 648A, v686∆TRS-TRL, v686 mut1, or v686 mut2 challenged group in bivalent-vaccinated birds is in Figure S7.

Figure 4

Proportional distances of the virulence of v686 mut1, v686 mut2, v686∆TRS-TRL, and the reference MDV strains with known pathotypes. The reference MDV strains are JM/102W (vMDV), Md5 (vvMDV), and 648A (vv+MDV).