IDENTIFICATION, CHARACTERIZATION, AND VALIDATION OF GENETIC MUTATIONS INCURRED DURING IN VITRO ATTENUATION OF MAREK'S DISEASE VIRUS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 0220760
• Grant No.: 2010-65119-20505
• Cumulative Award Amt.: $375,000.00
• Proposal No.: 2009-01659
• Project Start Date: Jan 15, 2010
• Project End Date: Jan 14, 2014
• Grant Year: 2010
• Program Code: [92521]- Animal Health and Well-Being: Animal Health

Recipient Organization
MIDWEST AREA, AGRICULTURAL RESEARCH SERVICE
3606 EAST MT HOPE ROAD
EAST LANSING,MI 48823

Performing Department
(N/A)

Non Technical Summary
Poultry is the third largest agricultural commodity and primary meat consumed in the U.S. In 2007, the U.S. produced 49.2 billion pounds of chicken meat, 7.87 billion pounds of turkey meat, and 90.6 billion eggs for combined sales totaling $31.9 billion, and the industry is the largest producer and exporter of poultry meat in the world. Several major issues confront the poultry industry today. With high-density chicken rearing and reduced genetic diversity from industry consolidation, control of infectious diseases and preventing disease outbreaks are critical for sustaining economic viability, maintaining public confidence in poultry products, and enhancing animal welfare. Among diseases, MarekOs disease (MD), a lymphoproliferative disease of poultry caused by the highly oncogenic a-herpesvirus Marek's disease virus (MDV), continues to be at or near the top of the list. The main control strategy for MD is vaccination. The first U.S. vaccine was HVT, a related herpesvirus of turkey, introduced in the late 1960s. Since then, additional vaccines with better efficacy have been introduced. While these vaccines are very effective in preventing tumor formation, they are not sterilizing, thus, do not prevent infection or shedding of virus. Based on history, a new MD vaccine is useful for about 10 years. With no new conventional vaccines in development, it appears likely that another major MD outbreak will occur in the near future. MD vaccines are produced by blind passages of field viruses in vitro. This process takes advantage of the well-documented knowledge that MDV mutates to less virulence during successive in vitro passages. While very effective, the molecular basis of this attenuation is unknown. It is very likely that changes of viral virulence are associated with viral genomic changes. Thus, it is highly desirable to know what genetic changes are responsible as this could lead to the rational design of more effective MD vaccines. Efforts to elucidate the molecular basis for changes in virulence are greatly hampered by the strictly cell-associated nature of the virus and they are mixed populations. With respect to trying to associate overall virulence and other MD-associated phenotypes, to date, no one has been able to associate them to specific DNA sequences (genotypes). Thus, for in vitro attenuated strains, even when the DNA sequence is determined, one can only compare it to the consensus sequence. In short, lack of information on specific genetic changes of the MDV genome confounded by mixed populations inhibit progress on understanding the molecular basis for the shift of MDV virulence and attenuation and, ultimately, the rational design of new MD vaccines. In this proposal, we apply new cutting-edge technologies that we believe can overcome the inability to identify genetic changes that occur in the MDV genome during in vitro attenuation. The novel use of genomic technologies will greatly aid in the rational development of new MD vaccines that have the potential to control new emerging and highly virulent MDV strains.

Animal Health Component

20%

Research Effort Categories

Basic

80%

Applied

20%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3210	1101	50%
311	3220	1101	50%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3210 - Egg-type chicken, live animal; 3220 - Meat-type chicken, live animal;

Field Of Science
1101 - Virology;

Keywords

marek's disease virus

vaccines

genetic variation

sequence

attenuation

Goals / Objectives
Marek's disease (MD) is one of the most serious chronic threats to the U.S. poultry industry due to recurring yet unpredictable vaccine breaks. Genetic mutations in Marek's disease virus (MDV), the causal pathogen, play a pivotal role in both the evolution of field strains with higher virulence and in vitro attenuation, the process used to make traditional vaccines. Unfortunately, little is known about naturally-occurring MDV mutations and their influences on virulence. This knowledge gap is attributed to the difficulty in obtaining genetically homogeneous virus stock as MDV is highly cell-associated. Consequently, MDV strains and vaccines are a mixed population of uncharacterized viral genomes. Recently, infectious molecular clones of MDV have become available, which provide defined homogenous populations. In this proposal, we utilize these MDV clones and next generation sequencing technologies to identify, monitor, and characterize viral mutations during the in vitro attenuation process. Our objectives are to: 1) Identify and correlate specific genetic and functional changes in the MDV genome that occur during the in vitro attenuation process with virulence, and 2) Experimental confirmation of the relevance of the data obtained in Objective 1 to the attenuation process. Answers to these fundamental questions should define the parameters for the molecular characterization of MDV strains, help promote the rational design of superior MD vaccines, provide molecular markers of MDV pathotypes, and aid in the prediction of MDV evolution in the field.

Project Methods
For objective 1, we take advantage of Md5B40BAC1, our BAC clone that generates virulent MDV. This homogeneous viral strain will be passaged in vitro for up to 80 times with 3 replicates. The virulence of the passaged populations will be tested by inoculating 500 pfu in MD susceptible chicks. To aid in the determination of what percent of the viral genomes are virulent or avirulent, we will also challenge additional sets of birds with 500 pfu of defined mixtures of cloned virulent and avirulent MDVs. The attenuated populations at the lowest passage level will be grown, the viral DNA enriched, and sequenced on an Illumina GA platform. Assuming the viral DNA comprises 33% or more of the sample, then the expected DNA sequence will provide ~500X coverage. The aligned sequences from each attenuated replicate will be compared with the Md5B40BAC1 sequence at the nucleotide level to identify polymorphisms, and the allele frequency of each mutation quantified. To help in the functional analysis of candidate mutations, we will also profile the viral RNA by sequencing it as well, which will also help to confirm polymorphisms revealed in the viral DNA. Analysis of the RNA sequencing should help to identify genetic changes that result in transcriptional or primary amino acid changes in specific MDV genes. Armed with information on which genes and polymorphisms might be necessary or sufficient for MDV attenuation, we will further test these candidates by determining their frequency during the in vitro passages using pyrosequencing. By doing so, we should be able to identify the genetic changes that exhibit the same kinetics as attenuation and, thereby, eliminating those not involved due to genetic drift. For objective 2, once we are able to narrow down candidate mutation(s) and gene(s) responsible for the attenuation, we will experimentally test them by generating defined recombinant MDV.

Progress 01/15/10 to 01/14/14

Outputs
Target Audience: Academics and scientists in biologics companies working in poultry diseases and vaccine production. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? A graduate student was trained in designing and conducting experiments, learning wet-lab computational biology, and reporting the results. How have the results been disseminated to communities of interest? Yes, through the scientific literature and public presentations What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Marek’s disease (MD) is a lymphoproliferative disease of chickens caused by the oncogenic Gallid herpesvirus 2, commonly known as Marek’s disease virus (MDV). MD vaccines, the primary control method, are often generated by repeated in vitro serial passage of this highly cell-associated virus to attenuate virulent MDV strains. To understand the genetic basis of attenuation, we used experimental evolution by serially passing three virulent MDV replicates generated from an infectious BAC clone. All replicates became completely or highly attenuated, indicating de novo mutation and not selection amongst quasi-species existing in a strain is the primary driving force for the reduction in virulence. Sequence analysis of the attenuated replicates revealed 41-95 single nucleotide variants (SNVs) at 2% or higher frequency in each population, and several candidate genes containing high frequency, nonsynonymous mutations. Five candidate mutations were incorporated into recombinant viruses to determine their in vivo effect. SNVs within UL42 (DNA polymerase auxiliary subunit) and UL46 (tegument) had no measurable influence, while two independent mutations in LORF2 (a gene of unknown function) improved survival time of birds but did not alter disease incidence. A fifth SNV located within UL5 (helicase-primase subunit) greatly reduced in vivo viral replication, increased survival time of birds, and resulted in only 0-11% disease incidence. This study shows that multiple genes, often within pathways involving DNA replication and transcriptional regulation, are involved in de novo attenuation of MDV and provides targets for the rational design of future MD vaccines.

Publications

Progress 01/15/11 to 01/14/12

Outputs
OUTPUTS: Attenuation of Mareks disease virus (MDV) can occur after many consecutive passages in tissue culture, and serves as the basis for the production of vaccines against Mareks 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 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 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 ~40%, ~60%, ~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). Although this was only a single trial, if this result is confirmed, it would demonstrate that a specific SNP in UL5 found in in vitro attenuated MDVs can impact the virulence of the virus. PARTICIPANTS: Evin Hildebrandt and Hans Cheng conducted the experimental and bioinformatic analyses. The Michigan State University Research Technology Support Facility conducted the DNA and RNA sequencing using an Illumina GA. TARGET AUDIENCES: People interested in our research include academic and industry scientists working on molecular virology, vaccine production, and poultry diseases. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
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 preliminary results support this contention as a single mutation in UL5 significantly reduces the virulence of the virus. 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.

Publications

• No publications reported this period

Progress 01/15/10 to 01/14/11

Outputs
OUTPUTS: Attenuation of Marek's disease virus (MDV) can occur as the result of many consecutive passages in tissue culture, and serves as the basis for the production of vaccines against Marek's disease (MD). While the process of attenuation is repeatedly observed, the genetic basis behind it is unknown. 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 BAC clone was used as the parental viruses to infect cultured chicken cells then serially passed for over 100 passages. To determine when attenuation occurred, the three replicates after 50, 60, and 70 passages were used to infect chickens with 500 pfu of the appropriate virus. MD incidence was determined via necropsy after 8 weeks, as well as in birds that died before 8 weeks, in order to identify the lowest passage number that is completely attenuated in each of the B40BAC replicates. 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 ~40%, ~60%, ~80% in the viral population, including one SNP which was fixed 100% in a replicate. PARTICIPANTS: Evin Hildebrandt and Hans Cheng conducted the experimental and bioinformatic analyses. The Michigan State University Research Technology Support Facility conducted the DNA and RNA sequencing using an Illumina GA. TARGET AUDIENCES: People interested in our research include academic and industry scientists working on molecular virology, vaccine production, and poultry diseases. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
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. One particular gene of interest that appears to fit that description and looks to be an excellent candidate involved in attenuation 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.

Publications

• No publications reported this period