Location: Endemic Poultry Viral Diseases Research
2018 Annual Report
Objectives
Objective 1: Characterize the evolution of avian tumor viruses in poultry production systems, including characterizing the effect of vaccination on the evolution of Marek’s disease virus field strains.
Sub-objective 1.1: Characterize the effect of vaccination on the evolution of Marek’s disease virus field strains.
Sub-objective 1.2: Surveillance for virulent strains of avian tumor viruses in field flocks and development of improved diagnostics for new strains.
Objective 2: Identify host-pathogen interactions that drive the transmission of avian herpesviruses, including identifying viral determinants that drive transmission and determining host genetic effects on virus transmission.
Sub-objective 2.1: Host and virus gene expression patterns in the skin cells facilitate production of cell-free enveloped infectious virus particles.
Sub-objective 2.2: Determine host genetic effect on virus transmission.
Objective 3: Elucidate the genetic and biological mechanisms that drive Marek’s disease resistance, including characterizing and defining innate defense mechanisms that contribute to Marek’s disease resistance.
Sub-objective 3.1: Role of the innate defense mechanisms that drive Marek’s disease resistance, including defining and characterizing innate defense mechanisms that contribute to Marek’s disease resistance.
Sub-objective 3.2: Define innate defense mechanisms that contribute to Marek’s disease vaccinal synergy.
Objective 4: Discover safe and highly effective vaccine platforms that convey protection against avian herpesviruses, including developing a vaccine strain of Marek’s disease virus serotype 1 that is cell free and does not require liquid nitrogen for storage and shipment, and discovering novel Infectious laryngotracheitis virus (ILTV) vaccine platforms that are safe, efficacious, and cost-effective.
Sub-objective 4.1: Develop cell-free Marek’s disease vaccine.
Sub-objective 4.2: Generate novel infectious laryngotracheitis virus vaccines.
Approach
Marek’s disease (MD) and infectious laryngotracheitis (ILT) are agronomically-important diseases of chickens caused by two alphaherpesviruses, Marek’s disease virus (MDV) and infectious laryngotracheitis virus (ILTV), respectively. Although chickens have been vaccinated against these diseases for decades and though highly successful, the vaccines fail to protect against reinfection and transmission. One significant consequence has been the evolution of more virulent MDV field strains in MD-vaccinated flocks. This vicious cycle of virus evolution followed by introduction of new expensive vaccines is not sustainable in the large, expanding, and highly concentrated chicken meat and egg industries. Another shortcoming of MD vaccination is the requirement for storage and transportation of viable vaccine virus in liquid nitrogen. These vaccines are prone to breaks in vaccine control due to improper handling and have restricted usage on a global basis due to the limits of cold chain processes in developing countries. Since current vaccines fail to induce complete immunity, we plan on investigate the role of innate immunity in preventing MDV infection, identify host and virus determinants involved in transmission that undoubtedly play a role in virus evolution, and define the mechanism by which MDV vaccine strains act synergistically in protective immunity. ILTV vaccines are also imperfect and recent research suggests that not only can they revert to virulence by simple bird-to-bird transmission, but also vaccine strains can recombine to generate new virulent strains. There is a need to engineer better modified-live ILT vaccines incapable of reversion to virulence and subunit vaccines incapable of recombination.
Progress Report
Substantial progress was made on all objectives of the project. A brief description of selected accomplishments is listed below. This year, we are comparing two methods of back passages of Marek’s disease virus (MDV) to determine the most efficient method for simulating virus evolution. One is by selection of isolated virus from birds with most significant pathology; the second is by natural transmission to contact birds.
For our virus transmission studies, we defined minimal exposure time for successful MDV transmission. The experimental design included donor birds challenged with Marek’s disease virus, which at 3 weeks post-challenge were placed with newly hatched recipient birds. Donor birds were either MD-resistant (Line 6) or MD-susceptible (Line 7). A portion of the recipient birds were removed and transferred to new isolators during the following times post-exposure to donor birds: 4, 8, 12, 24, 48, 96 and 168 hours. Transferred recipient birds were monitored for 8 weeks and necropsied to determine whether they developed MD. Results suggested that the minimal exposure time of 4 hours was sufficient for successful MDV transmission from both donor lines.
Also this year, we addressed the duration of the infectious period of the donor birds. Experiments were completed with donor birds that were either MD-susceptible (Line 7, Line 15.P-19) or MD-resistant (Line 6, Line 15.N-21), challenged with MDV. After challenge, donor birds were transferred to new isolators of naïve recipient birds on days 4, 8, 12, 16, and 20, followed by weekly transfers at days 28, 35 and 42. Recipient birds were monitored for 8 weeks and necropsied to determine whether they developed MD. Each donor bird was sampled at transfer and each recipient bird was bled at 7 and 14 days post-exposure to donor birds. Results demonstrated initiation of transmission beginning at days 12, 16 or 20 depending on the donor line of bird and trial. All of the later time points were successful for virus transmission, so there was no beginning and end of the infectious period.
To study the phenomenon of Marek’s disease virus vaccine competition, we completed protection studies using pairs of rHVT vaccines administered together followed by inoculation with Newcastle disease virus (NDV), infectious bursal disease virus (IBDV), or infectious laryngotracheitis virus (ILTV). HVT vector vaccines with ILTV or IBDV gene insertions were more susceptible to interference compared to NDV insertion. Interference was not related to differences in virus replication of each vector.
The ability of two Marek’s disease (MD) vaccines to surpass protection that either individual vaccine alone can provide against Marek’s disease virus (MDV)-induced pathology is called vaccinal synergism. Synergism using two vaccines has been widely used since the early 1980s. Despite this widespread and long usage, the mechanism of how MD vaccinal synergy provides superior protection has never been elucidated. To elucidate the underlying mechanism, we have characterized chickens vaccinated with either a single MD vaccine (monovalent) only or both (bivalent). We confirm that bivalent MD vaccines provide higher protection compared to either monovalent MD alone. Our results also indicate that bivalent vaccines limit MDV replication up to 8 weeks while both monovalent MD vaccines failed to limit MDV replication. We also find that the replication of monovalent vaccines differ with respect to time after administration and organ. These results suggest that bivalent vaccines work additively by replicating in different tissues and at different times to promote different types and extended periods of immune responses against MDV. Our current and future studies should provide valuable insights on the mechanisms of MD vaccine synergy and MD vaccines, in general.
Significant progress has been made towards the generation of a two vector system for the reconstitution of infectious laryngotracheitis. The generation of ILTV mutants have traditionally been difficult due to low frequency of recombination using marker rescue experiments and the lack of an infectious clone or bacterial artificial chromosome (BAC). Previously, we have been able to reconstitute ILTV from a collection of cosmids and yeast centromere plasmids (ycp). Although three recombinants have been generated using this methodology, this methodology is cumbersome. To alleviate issues regarding low homologous recombination efficiencies and low transfection efficiencies, a large ycp vector was created by connecting the inserts from the three recombinant cosmids (cos34, cos28 and cos27) into the ycp recombinant (1A), which contained the 3’ end of the US, the terminal repeat short and the 5’ end of the unique long. Addition of this construct along with cosmid 52 should reconstitute the virus upon transfection of LMH cells and generate a platform for the incorporation of genes encoding other antigens or immune stimulating proteins.
A bioinformatics analysis of full genome sequences of Marek’s disease viruses has revealed two independent pathways that lead to virulence in Eurasia and North America. It has been known that the severity of MDV infection in chickens has been rising steadily since the adoption of intensive farming techniques and vaccination programs in the 1950s and 1970s respectively and the emergence of virulent viruses appears to coincide approximately with the introduction of comprehensive vaccination on both continents. Interestingly, our examination of gene-linked mutations could not identify a strong association between mutational variation and virulence phenotypes, indicating that MDV may have evolve readily and rapidly under strong selective pressures, and that multiple genotypic pathways may underlie virulence adaptation in MDV.
Phylogenetic and comparative genomic studies of infectious laryngotracheitis viruses has mainly focused on the differences among attenuated vaccines, vaccinal revertants and natural recombinant strains from Australia, the United States, and China. This analysis suggests the majority of isolates have a close relationship with the vaccine viruses and are largely considered vaccinal revertants. In a search to identify wild-type parental strains, the genomes of 5 ILTV isolates from backyard and commercial flocks in the U.S. were sequenced using the MiSeq technology. In comparison with other complete ILTV genomes in GenBank, a diagnostic assay was developed to differentiate strains based on single nucleotide polymorphisms (SNPs) in the loci encoding ORF A and ORF B. In a blind study, a 90% correlation between the single locus assay and the conventional multi-locus assay was achieved. The single locus assay can be performed using classical Sanger sequencing and the third generation sequencing (TGS) technology Minion. Interestingly the TGS technology was demonstrated to be more sensitive then Sanger due to the incorporation of a nested polymerase chain reaction (PCR) step.
The genome of GaHV-3 strain 301B/1 was determined using next-generation sequencing technology (Illumina’s MiSEQ) and a consensus sequence was generated. Seventeen BAC clones of GaHV-3 that contained the entire full genome were constructed and characterized using restriction endonuclease profiling. Infectious 301B/1 viruses from selected BAC clones were recovered by reverse genetics technology and their growth kinetics determined. The in vitro characteristics of reconstituted GaHV-3 strain 301B/1, derived from BAC clones, indicated that they grew to similar titers as wildtype virus. Two candidate recombinants were chosen for further in vivo experiments. In vivo efficacy studies using the two reconstituted viruses (GaHV-3 strain 301B/1 A and B) as vaccines indicated protection against very virulent Marek’s disease (strain Md5) challenge with protective indices of approximately 80%. This indicate a level of protection comparable to currently used SB-1 vaccine stains against MD.
Accomplishments
1. Minimal exposure time for Marek’s disease virus transmission. For the purpose of determining whether genetic selection can be used to reduce Marek’s disease virus transmission, ARS researchers in East Lansing, Michigan, completed initial studies to evaluate minimal exposure time for successful Marek’s disease virus transmission. The experimental design included donor birds challenged with Marek’s disease virus, which at 3 weeks post-challenge were placed with newly hatched recipient birds. Donor birds were either MD-resistant (Line 6) or MD-susceptible (Line 7). A portion of the recipient birds were removed and transferred to new isolators during the following times post-exposure to donor birds: 4, 8, 12, 24, 48, 96 and 168 hours. Transferred recipient birds were monitored for 8 weeks and necropsied to determine whether they developed MD. Results suggested that the minimal exposure time of 4 hours was sufficient for successful MDV transmission from both donor lines.
2. Duration of infectious period for Marek’s disease virus. For the purpose of determining whether genetic selection can be used to reduce Marek’s disease virus transmission, ARS researchers in East Lansing, Michigan, addressed the duration of the infectious period of the donor birds. Experiments were completed with donor birds that were either MD-susceptible (Line 7, Line 15.P-19) or MD-resistant (Line 6, Line 15.N-21), challenged with MDV. After challenge, donor birds were transferred to new isolators of naïve recipient birds on days 4, 8, 12, 16, and 20, followed by weekly transfers at days 28, 35 and 42. Recipient birds were monitored for 8 weeks and necropsied to determine if they developed MD. Each donor bird was sampled at transfer and each recipient bird was bled at 7 and 14 days post-exposure to donor birds. Results demonstrated initiation of transmission beginning at days 12, 16 or 20 depending on the donor line of bird and trial. All of the later time points were successful for virus transmission, so there was no beginning and end of the infectious period.
3. Turkey herpesvirus (HVT) has been widely used as a vaccine for Marek’s disease (MD) since the 1970s. Because HVT is a safe vaccine that is poorly sensitive to interference from maternally derived antibodies, it has seen rising use as a vector for vaccines by the insertion of antigenic genes from poultry viruses such as Newcastle disease virus (NDV), infectious bursal disease virus (IBDV) and infectious laryngotracheitis virus (ILTV). These recombinant HVT vector (rHVT) vaccines have been shown to offer similar protection against MDV challenge compared to standard HVT vaccination, however, it has been suggested that different rHVT products cannot be combined with each other or with standard HVT due to interference among HVT strains. ARS researchers at East Lansing, Michigan, compared virus replication kinetics for individual HVT and rHVT vaccine strains both in vitro and in vivo and found significant differences between strains. Protection studies demonstrated that HVT vector vaccines with ILT or IBD gene insertions were more susceptible to interference compared to NDV insertion. Interference was not related to differences in virus replication of each vector. This confirmed phenomenon will provide the basis for additional studies to understand the mechanism behind competition and synergism of Marek’s disease vaccine strains.
4. The modified live vaccines against infectious laryngotracheitis are protective and can reduce virus shedding, however they can revert to virulence and infect unvaccinated birds. To prevent this, ARS researchers at Athens, Georgia, sought to develop a molecular clone of ILTV. Previously, four overlapping cosmid clones and a yeast centromere plasmid (ycp) clone that contain large fragments of the ILTV genome were generated and sequenced. Reconstituted viable virus could be recovered when LMH cells were transfected with these 5 clones and ancillary plasmids encoding transactivating proteins. To simplify this method we have combined the inserts from three of the cosmid clones into the ycp recombinant to generate a large 134 kilobase construct. This large construct and only one other cosmid clone (cos52) have been used to generate plaques in transfected LMH cells. This two vector system to reconstitute ILTV can be easily manipulated in vitro to generate vaccine strains as well as vaccines containing multiple antigens.
5. Genotyping of ILTV strains is laborious and costly. Often multiple genes have to be sequenced following PCR amplification in order to identified meaningful allelic variations. Furthermore, diagnostic laboratories across the globe use different loci so there is no standardized genotyping method for ILTV. To simplify this, ARS researchers at Athens, Georgia, analyzed all the full genome ILTV sequences in GenBank and identified 6 single nucleotide polymorphisms (SNPs) within a single locus that can differentiate the strains into 4 genotypes. A simple PCR-based method was developed using a single pair of primers, and the sequencing of the PCR products is amendable to both dideoxy (Sanger) sequencing as well as third generation sequencing technology based on nanopore sequencing (MinION). This assay is highly sensitive with a short turnaround time and can be multiplexed with other DNA/cDNA-based diagnostic assays when barcoded primers are included.
6. Novel vector vaccine platform based on strain 301B/1 of non-oncogenic Gallid herpesvirus 3 (GaHV-3) to protect against Marek’s disease. ARS researchers in Athens, Georgia, developed a novel vector vaccine platform based on strain 301B/1 of non-oncogenic Gallid herpesvirus 3 (GaHV-3) to protect against Marek’s disease. This platform is extremely versatile and can incorporate genes encoding antigens of other poultry pathogens. The 301B/1 strain has been demonstrated to work synergistically with the widely-used Turkey Herpesvirus (HVT) vaccine. The genomic sequence of strain 301B/1 was determined using next-generation sequencing (MiSEQ) with DNA purified from virus capsids. Overall the genome structure is very similar (99% identity) to the SB-1 vaccine strain of GaHV-3, but unlike SB-1 virus, no avian retrovirus sequences (known as LTR sequences) were found in the 301B/1 genome.
7. The entire genome of GaHV-3 strain 301B/1 was molecularly cloned into a Bacterial Artificial Chromosome (BAC) plasmid by ARS scientists in Athens, Georgia. Two reconstituted 301B/1 viruses from BAC clones were characterized in vitro and further examined in vaccine protective efficacy studies against pathogenic Marek’s disease virus challenge. The two BAC-derived 301B/1 viruses had comparable protection efficacies. The resulting BAC clones are valuable tools in an arsenal of reagents, developed by ARS researchers at Athens, Georgia, that allow rapid and precise site-directed modifications or recombineering of viral genomes in order to develop efficacious vector vaccines not only against Marek’s disease but against a plethora of other important poultry diseases.
Review Publications
Conrad, S.J., Silva, R.F., Hearn, C.J., Climans, M., Dunn, J.R. 2018. Attenuation of Marek’s disease virus by codon pair deoptimization of core herpesvirus genes. Virology. 516:219-226. https://doi.org/10.1016/j.virol.2018.01.020.
Conrad, S.J., Silva, R.F., Hearn, C.J., Climans, M., Dunn, J.R. 2018. Attenuation of Marek's disease virus by codon pair deoptimization of a core gene. Virology. 516:219-226. https://doi.org/10.1016/j.virol.2018.01.020.
Xie, Q., Chang, S., Dong, K., Dunn, J.R., Song, J., Zhang, H. 2017. Genomic fariation between genetic lines of white leghorns differed in resistance to Marek’s disease. Journal of Clinical Epigenetics. 3:29. https://doi.org/10.21767/2472-1158.100063.
Webb, A.E., Youngworth, I.A., Kaya, M., Gitter, C.L., O’Hare, E.A., May, B.P., Cheng, H.H., Delany, M.E. 2018. Narrowing the wingless-2 mutation to a 227 Kb candidate region on chicken chromosome 12. Poultry Science. 97(6):1872–1880. https://dx.doi.org/10.3382/ps/pey073.
Warren, W.C., Hillier, L.W., Tomlinson, C., Minx, P., Kremitzki, M., Graves, T., Markovic, C., Bourk, N., Pruitt, K.D., Thibaud-Nissen, F., Schneider, V., Mansour, T.A., Brown, C.T., Zimin, A., Hawken, R., Abrahamsen, M., Black Pyrkosz, A.A., Morrison, M., Fillon, V., Vignal, A., Chow, W., Howe, K., Fulton, J.E., Miller, M.M., Lovell, P., Mello, C.V., Cheng, H.H. 2017. A new chicken genome assembly provides insight into avian genome structure. G3, Genes/Genomes/Genetics. 7(1):109-117. https://doi.org/10.1534/g3.116.035923.
Trimpert, J., Groenke, N., Jenckel, M., He, S., Kunec, D., Szpara, M.L., Spatz, S.J., Osterrieder, N., Mcmahon, D.P. 2017. A phylogenomic analysis of Marek’s disease virus reveals independent paths to virulence in Eurasia and North America. Evolutionary Applications. 10(10):1091-1101. https://doi.org/10.1111/eva.12515.
