Attenuation of a very virulent Marek's disease herpesvirus (MDV) by codon pair bias deoptimization

Figures

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  Fig 1. Distribution of codon pair bias (CPB) scores and recoding of the MDV gene UL30. (A) Distribution of calculated codon pair bias (CPB) scores of 15,762 predicted chicken, 112 MDV, and recoded MDV UL30 genes. Each light blue circle represents a calculated CPB score of a single chicken protein coding gene plotted against its protein length (amino acids). The arithmetic mean of all 15,762 CPB scores is 0.0755. The blue diamonds represent 112 predicted MDV protein coding genes. The pink circle represents original, wild type, labeled WWW, MDV UL30 gene. RWW, RRW and RRR (yellow crosses), which overlap each other and the parental UL30 represent calculated CPB scores of recoded MDV UL30 genes that have one, two or three segments of the ORF codon pairs-randomized. The red diamonds and green squares represent recoded UL30 genes that have either first, first two or all three segments of UL30 ORF codon pair-deoptimized (DWW, DDW, DDD) or optimized (OWW, OOW, OOO). (B) Structure of the MDV UL30 genomic region. UL30 encodes the catalytic subunit of the DNA polymerase, UL31 encodes a nuclear egress protein. UL30 and UL31 overlap at their 3’ ends by 98 nucleotides. UL30 is 3,663 nucleotide long…

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  Fig 2. Protein production from differently recoded UL30 genes. Representative images of HEK 293T co-transfected with plasmid expressing mKate2 and recoded UL30-EGFP fusion genes. Note that EGFP in UL30-EGFP fusions can be found primarily in the nucleus because UL30 encoding DNA Pol contains a nuclear localization signal, but EGFP produced by the control plasmid (pEGFP-N1) is primarily present in the cytoplasm of transfected cells. Cells were imaged on a Zeiss Axiovert S100 microscope 24 h post transfection at 400-fold magnification.

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  Fig 3. Quantification of RNA expression and protein production from the recoded UL30 genes. HEK 293T cells were transfected with dual expression plasmids pVITRO2-TagBFP-UL30-EGFP that carried differently recoded UL30 genes fused in frame with EGFP gene. 24 h post transfection RNA expression (A) from the recoded genes was quantified by qPCR, and protein production by flow cytometry (B). The UL30 RNA levels were normalized against the TagBFP levels. We used EGFP fluorescence as a reporter to quantify protein production of the fusion UL30-EGFP genes. The EGFP fluorescence was normalized against the TagBFP fluorescence. P-values were calculated using Kruskal-Wallis H test, * indicates P<0.05.

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  Fig 4. Characterization of recoded MDV UL30 mutants. (A) Effect of recoding on UL30 expression from the virus background. CEC were transfected with the parental or mutant BAC clones that carried differently recoded UL30 genes. 24 h post transfection RNA levels of UL29, UL30 and UL42 genes were quantified by qPCR. P-values were calculated using Kruskal-Wallis H test, * indicates P<0.05. (B) Plaque sizes of the parental and mutant viruses in CEC cells 6 days post infection. All images were taken at 100-fold magnification using an inverted fluorescence microscope. The box-plot displays the distribution of relative plaque diameter normalized against the average plaque diameter of the parental virus. P-values were calculated using one-way ANOVA Bonferroni’s multiple comparison test, *** indicates P<0.001. (C) Representative images of plaques formed by parental and mutant viruses. Plaque produced by the vOOO virus were larger, and plaques formed by the vDDW and vWDD viruses were markedly smaller than those formed by the vWWW virus. Scale bar, 200 μm.

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  Fig 5. Multi-step growth kinetics of indicated viruses shown as mean and SEM. 1×106 CECs were infected with 100 PFU and viral progeny was titered 1–6 days post infection. (A) Comparison of growth curves of the parental (vWWW) and mutant viruses (vRRR, vOOO, vDWW and vDDW); n = 6, Kruskal-Wallis H test, * indicates P<0.05. (B) Comparison of growth curves of the parental (vWWW) and revertant viruses (vRRR-Rev, vOOO-Rev, vDWW-Rev and vDDW-Rev); n = 6, Kruskal-Wallis H test, P<0.05.

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  Fig 6. Replication of MDV in vivo. (A) Blood samples of chickens infected with the indicated viruses were taken at 4, 7, 10, 14, and 28 days post infection. (B) Contact chickens were sampled 14, 21, 28, 35 and 42 days p.i. Viral titers in the blood are shown as MDV genome copy numbers per 1×106 cells of eight infected chickens per group. The detected viral loads are not statistically different among the groups, Kruskal-Wallis H test.

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  Fig 7. Codon pair deoptimization of UL30 impairs disease development and tumor formation in vivo. (A) MD incidence in chickens infected with the parental (vWWW), mutant (vOOO, vDWW, vDDW) and revertant (vOOO-Rev, vDWW-Rev, vDDW-Rev) viruses. Animals infected with the vDDW mutant showed lower MD incidence than those that were infected with the parental virus. Comparison of survival curves via Log-rank (Mantel-Cox) test did not identify statistical differences between the groups. Tumor incidence in infected chickens (B) and contact sentinel chickens that were housed together with the infected chickens (C). Chickens that were infected with three revertant viruses were housed together in one room and shared one group of sentinel chickens. Tumor incidence is shown as the percentage of animals per group. Differences in tumor incidence among the groups of infected chickens are statistically significant. Tumor formation was impaired in contact chickens that were housed together with vDWW or vDDW infected chickens. Statistical analysis was done by Chi-square test, P<0.0001.