2013 Annual Report
1a.Objectives (from AD-416):
Marek’s disease virus (MDV), unlike most other viruses, is highly cell-associated and consequently, exists as population rather due to the inherent process of mutations. This makes it very difficult to determine the relationship between genetic variation and phenotypic variation. With the advent of molecular clones of MDV-BAC clones and genomic technologies, we can now better address how specific genetic changes influence virus virulence, vaccine efficacy, etc. Specifically, we would like to know during cell passage, are there preferred sites that are required for MDV attenuation. The proposed experiments will guide scientists on how to molecular characterize MDV field strains, what changes occur during cell passage attenuation and vaccine production, and other fundamental knowledge.
1b.Approach (from AD-416):
Our virulent BAC cloned MDV genome that generates a fully virulent virus will be passed in cell culture, which is known to attenuate the virus. At every 10 passages, the viral population will be used to challenge chickens to determine the amount of MD incidence. This will continue until the viral population is completely avirulent. Preceeding populations will have their viral genome purified and sequenced using next generation sequencers (e.g., Illumina GA or ABI SOLiD) to identify polymorphisms in the genome as well as the allele frequency. In addition, RNAs from the same sequenced populations will be sequenced, which when combined with the genomic sequence information, will confirm polymorphisms and reveal changes in viral gene transcription pattern. Following analysis, key genetic changes will be introduced into the virulent viral genome to address whether the polymorphisms do promote attenuation.
This project is directly linked to Specific Cooperative Agreement 3635-32000-016-03S titled "Identification, Characterization, and Validation of Genetic Mutations Incurred During In Vitro Attenuation of Marek’s Disease Virus." Attenuation of Marek’s disease virus (MDV) can occur after many consecutive passages in tissue culture, and serves as the basis for the production of vaccines against Marek’s disease (MD). In order to better understand the precise genetic changes that occur during attenuation through repeated cell passages, this experiment intends to identify the specific genes that are altered. The virulent MDV bacterial artificial chromosome (BAC) clone was used as the parental viruses to infect cultured chicken cells then serially passed for over 100 passages. After 50, 60, and 70 passages, the resulting viruses were used to infect chickens with 500 plaque-forming unit (PFU) to determine virulence. Rates of attenuation varied between the three serially passed replicates. It was determined that complete attenuation occurred after 60 passages in Replicate 2 and 70 passages in Replicate 3, while Replicate 1 was still not completely attenuated after 70 passages (1/17 positive for MD). It was anticipated that Replicate 1 would be completely attenuated after 70 passages, yet later bird trials of Replicate 1 after 80 passages still resulted in 1/16 birds developing nerve enlargements and positive for MD after 8 weeks. Additionally, a dramatic decrease in MD incidence occurred very rapidly, in which the number of MD positive birds decreased from about 80-100% of birds to 0-24% within only 10 passages. The lowest attenuated passage was used for sequencing of both MDV DNA and RNA. The 75 base reads were trimmed and mapped to the MDV reference (HQ149525.1) and polymorphisms identified. Compared to the BAC reference, 41-95 SNPs occurred at >2% in the viral population, depending on the replicate. While identical nucleotide mutations were not shared among attenuated replicates, mutated genes containing non-synonymous mutations were in common among certain pairs of replicates. Genes MDV 017 and MDV096 contained non-synonymous mutations in both Rep 1 and 2, while RLORF4 and MDV 059 contained non-synonymous mutations in Reps 2 and 3 and MDV 088 contained non-synonymous mutations in both Rep 1 and Rep 3, although not all of the mutations are at high frequencies. The mutations of greatest interest within genes in common among all three attenuated lineages occurred in ICP4. Multiple non-synonymous mutations were found in ICP4 for all attenuated replicate lineages, of which some mutations were at frequencies at approximately 40%, approximately 60%, and approximately 80% in the viral population, including one SNP which was fixed 100% in a replicate. To test whether specific SNPs are responsible to attenuation, single mutations found in UL42 (DNA polymerase), UL45 (tegument), and UL5 (DNA helicase) were introduced. The UL42-containing mutant exhibited an altered in vitro phenotype, however, showed no reduction in the ability to induce disease or transfer horizontally. Similarly, the UL45 recombinant did not exhibit any altered phenotypes. On the other hand, the UL5 recombinant had a significant reduction in MD incidence (11% vs. 100% for the parental strain). This result has been confirmed in two additional trials where the UL5 recombinant virus lead to 0% MD incidence. Thus, we have demonstrated that a specific SNP in UL5 found in in vitro attenuated MDVs can impact the virulence of the virus. This study shows that an MDV BAC can be attenuated via in vitro passage due to serial passage generating de novo mutations during in vitro growth. Therefore, the process of serial passage yields new attenuated viruses through mutation, not just a process of selection for pre-existing viruses already better adapted for in vitro growth in order to cause attenuation of the strain. The dramatic decrease of MD incidence by nearly 80% within 10 passages also suggests that there might be a limited number of loci responsible for attenuation. Our results with the UL5 support this contention. Another gene of interest is ICP4. Considering that between 3-8 non-synonymous mutations are found in any attenuated replicate and many of those non-synonymous mutations occur at high frequencies in the attenuated replicates, these factors point towards ICP4 as a gene deserving further study. Additionally, it could be speculated that these mutations within ICP4 could affect the downstream expression of early and late genes regulated by mutations within this immediate early transcriptional regulator. This may be a possible explanation for the 2x increased expression of vIL-8 and UL45 in the attenuated replicates, despite those genes lacking mutations within the genes themselves.