Distinct polymorphisms in a single herpesvirus gene are capable of enhancing virulence and mediating vaccinal resistance

Abstract

Modified-live herpesvirus vaccines are widely used in humans and animals, but field strains can emerge that have a higher virulence and break vaccinal protection. Since the introduction of the first vaccine in the 1970s, Marek's disease virus overcame the vaccine barrier by the acquisition of numerous genomic mutations. However, the evolutionary adaptations in the herpesvirus genome responsible for the vaccine breaks have remained elusive. Here, we demonstrate that point mutations in the multifunctional meq gene acquired during evolution can significantly alter virulence. Defined mutations found in highly virulent strains also allowed the virus to overcome innate cellular responses and vaccinal protection. Concomitantly, the adaptations in meq enhanced virus shedding into the environment, likely providing a selective advantage for the virus. Our study provides the first experimental evidence that few point mutations in a single herpesviral gene result in drastically increased virulence, enhanced shedding, and escape from vaccinal protection.

Fig 1. Characterization of the recombinant viruses in vitro.

(A) A schematic illustration of the evolution of MDV towards increased virulence in the context of the indicated vaccine generations. (B) The representation of the Meq protein with its domains. The N-terminal region comprises of a proline/glutamine (Pro/Gln) rich domain followed by the basic region and the leucine zipper (ZIP). (C) Virus replication was assessed by multi-step growth kinetics. Mean viral genome copies per one million cells are shown for the indicated viruses and time points (p>0.05, Kruskal–Wallis test, n = 3). (D) Plaque size assays of indicated recombinant viruses. The mean plaque diameters of three independent experiments are shown as box plots with minimums and maximums (p>0.05, one-way ANOVA, n = 150). (E) The meq expression levels in infected CEC were assessed by RT-qPCR. Meq expression is shown relative to one million copies of the cellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and were not statistically different (Kruskal-Wallis test). (F) RT-PCR analysis of the meq/vIL8 splice variant using primers specific for the donor site “D” in meq and the acceptor sites “A” in vIL8. GAPDH was used as a control.

Fig 2. Influence of meq isoforms from various pathotypes on MDV pathogenesis.

(A) MDV genome copies were detected in the blood samples of chickens infected with indicated viruses by qPCR. Mean MDV genome copies per one million cells are shown for the indicated time points (p>0.05, Kruskal-Wallis test). (B) Disease incidence in chickens infected with indicated recombinant viruses and significant differences in comparison to vvMeq (** p<0.0125, Log-rank (Mantel-Cox) test). (C) Tumor incidence as percentage of animals that developed tumors during the experiment. Asterisks indicate significant differences compared to vvMeq (* p<0.05 and ** p<0.0125; Fisher’s exact test). (D) Tumor distribution is shown as the number of tumorous organs in tumor-bearing animals with standard deviations (* p<0.05 and ** p<0.0125; Fisher’s exact test).

Fig 3. Pathogenesis and tumor incidence in naïve contact animals.

(A) qPCR analysis of blood samples from naive chickens where MDV genome copies were determined (p>0.05, Kruskal-Wallis test). (B) Disease incidence in naïve chickens infected via the natural route and tumor incidence (C) and tumor distribution (D) are shown for co-housed contact animals. Asterisks (** p<0.0125; Fisher’s exact test) indicate the significant differences in (C).

Fig 4. Pathogenesis and shedding of different meq isoform viruses in vaccinated chickens.

Viral genome copy numbers of (A) the meq isoform viruses and (B) the HVT vaccine detected in blood of vaccinated chickens infected with the meq isoform viruses (p>0.05, Kruskal-Wallis test). (C) Disease incidence and (D) tumor incidence in vaccinated chickens infected with indicated recombinant viruses. Asterisks (** p<0.0125, Fisher’s exact test) indicate statistical differences to vv+Meq in (D). (E) Viral copies from feathers of the meq recombinant viruses. (A), (B) and (E): mean MDV genome copies per one million cells are shown for the indicated time points. (F) Viral copies per μg of dust are shown for each group as validated previously [32]. Statistical differences in the feathers and dust samples are displayed as a comparison to vvMeq. Asterisks indicate significant differences (* p<0.05 and ** p<0.0125; Tukey's multiple comparisons test).

Fig 5. Efficiency of meq isoform viruses to overcome innate immune responses.

Primary T cells were activated by innate immunity agonists (Poly I:C, LPS, or cGAMP). Activated T cells were infected with the different meq isoform viruses to determine the effects on virus shedding and replication. (A) Plaque counts were performed on CEC overlaid with 1,000 activated infected primary T cells. (B) Corresponding changes in plaque sizes on infected CEC (normalized to vvMeq). Asterisks indicate significant differences (* p<0.05 and ** p<0.0125; Tukey's multiple comparisons test).