Location: Endemic Poultry Viral Diseases Research2017 Annual Report
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.
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.
Substantial progress was made on all objectives of the project. A brief description of selected accomplishments is listed below. We completed the first of a series of studies to evaluate viral loads in Marek’s disease virus (MDV)-inoculated donor birds and contact recipient birds exposed to the donor birds to study whether genetic selection can be used to reduce Marek’s disease virus transmission. Virus load was significantly higher in Marek’s disease (MD)-susceptible compared to MD-resistant donor birds by 3-4 weeks post challenge. Similarly, virus load in the recipient birds exposed to the MD-susceptible donor birds was significantly higher compared to the group exposed to MD-resistant donors. In addition, donor vaccination status was also compared, which demonstrated that virus load was far greater in unvaccinated donor birds compared to vaccinated donor birds. Recipient birds housed with the unvaccinated donor birds appeared to become infected earlier and with much higher virus load compared to recipients housed with vaccinated donor birds. To explain our previous findings that di-codon deoptimization of Marek’s disease virus genes can lead to reduced pathogenicity, we developed an assay to measure changes in gene expression using a dual-promotor plasmid with self-cleaving fluorescent tag, which confirmed reduced gene expression of the deoptimized genes. These results demonstrate that altering the di-codon bias of select herpesvirus genes can affect the pathogenicity of the virus and may provide a novel method for MDV vaccine attenuation. To study the phenomenon of Marek’s disease virus vaccine competition, we compared virus replication kinetics for individual turkey herpesvirus (HVT) and recombinant HVT (rHVT) vaccine strains both in vitro and in vivo and found significant differences between strains. Protection studies were then performed using pairs of rHVT vaccines administered together followed by inoculation with Newcastle disease virus, infectious bursal disease virus, or infectious laryngotracheitis virus. A loss of protective efficacy was demonstrated against some viruses following certain vaccine combinations. We have completed RNAseq-based host and viral gene expression profiling in the skin of MDV infected chickens and are in the process of analyzing our data. Additional work was done by comparing DMV gene expression pattern in the skin and spleen tissues of infected chickens. Differential expression of MDV in the skin in comparison to the spleen provided further insights into mechanism of viral pathogenicity in the skin that lacks a vigorous adaptive immune response that is essential for controlling viral infection and dissemination. As for the role of innate immune system in vaccine-induced protection, we have provided flow cytometric evidence that vaccination activates natural killer (NK) cells that induces significant up regulation of CD107a, an activation marker for NK cells. We have also practiced bursectomy (last year) and isoflurane-based anesthesia that is needed for surgical removal of bursa. Initial trial for bersectomy and antibody-mediated B and T cell depletion is scheduled for July-August, 2017. Additionally, we have produced sufficient anti CD4 and anti CD8 T cells antibodies using hybridoma cell lines and have tested their isotype and specificity in Western blots. Marek’s disease (MD) vaccinal synergy is a phenomenon where certain combinations of MD vaccines work much better than either vaccine alone. To help understand the underlying biological mechanisms, birds were vaccine with only one (monovalent, HVT or SB-1) or both (bivalent) commonly used MD vaccines. Analysis of viral replication rates and tissue tropisms revealed that the two vaccines had very different patterns, which was not influenced by the presence of the other vaccine. This suggests that the mode of action of each MD vaccine is unique to that viral strain, and that synergy may be the result of unique and additive host responses. Significant progress has been made towards the generation a bivalent vaccine for both infectious laryngotracheitis virus (ILTV) and Newcastle disease virus (NDV). The fourth phase of the NDV/ILTV project involved trying to rescue donor constructs containing NDV genes [i.e. fusion (F) and hemagglutinin-neuraminidase (HN)] into the genome of ILTV. Numerous attempts were made to use classical homologous recombination or “marker rescue” to rescue donor constructs containing NDV genes into the genome of ILTV. Briefly, ILT viral DNA, isolated from gradient purified virions, was added to Leghorn Male Hepatoma (LMH) cells along with the linearized donor constructs (plasmids containing the cloned NDV genes plus the green fluorescent protein marker) and support plasmids (encoding UL48 and ICP4). Although this procedure had been used by us in the past to generate knock out mutants within the Open Reading Frame C and gJ loci, no fluorescent green plaques were ever detected during the original transfections or subsequent passages. This was tried with two different transfection reagents and various amount of viral DNA/plasmids/transfection lipid in differing ratios. The end results were plaques containing wild type virus. There seemed to be a problem with the virus’s ability to recombine and was frustrating and disappointing. We changed our approach to instead focus the generation of ILTV recombinants through the usage of overlapping cosmid clones. We received four cosmid clones (cos 34, cos 28, cos 27 and cos 52) from an associate and characterized these clones by determining their nucleotide sequences. Unfortunately, these clones only contained sequences that covered approximately 80% of the ILTV genome. The 3’ end of the terminal short region and the 5’ end of the unique long region were missing. This “missing” region was ~20,000 nucleotides long and we subsequently cloned it into the yeast vector pYES1L. This missing piece along with the four other cosmids were linearized with unique restriction endonucleases that cleave in regions near vector sequences, or in ILTV insert regions that are unimportant for homologous recombination. After linearization these pieces were used in transfection experiments with LMH cells. Infectious virus was finally recovered from the transfected cells and this is a major breakthrough since reconstitution of ILT virus from recombinant clones (e.g. cosmids, Bacteria Artificial Chromosome, Yearly Artificial Chromosome, etc) has never been reported. This achievement was not easily done since growth conditions of the E.coli harboring the cosmids had to be optimized. In vitro growth experiments demonstrated that the virus reconstituted from overlapping had growth kinetic practically identical to the of the virulent USDA reference strain of ILTV. In vivo pathogenicity studies in SPF birds indicate that the reconstituted recombinant virus is virulent to a similar degree as parental wild type virus.
1. Validation of virus transmission sampling methods. For the purpose of determining whether genetic selection can be used to reduce Marek’s disease virus (MDV) transmission, ARS researchers in East Lansing, Michigan completed the first of a series of studies to evaluate viral loads in MDV-inoculated donor birds and contact recipient birds exposed to the donor birds. Virus load was significantly higher in Marek’s disease (MD)-susceptible compared to MD-resistant donor birds by 3-4 weeks post challenge. Similarly, virus load in the recipient birds exposed to the MD-susceptible donor birds was significantly higher compared to the group exposed to MD-resistant donors. Given the identical genetic background of the recipient birds, this indicates that either the MD-susceptible donor birds may have exposed the recipient birds slightly earlier, or may have exposed the recipient birds to a higher dose of initial infection compared to the MD-resistant donor group. In addition, donor vaccination status was also compared, which demonstrated that virus load was far greater in unvaccinated donor birds compared to vaccinated donor birds. Recipient birds housed with the unvaccinated donor birds appeared to become infected earlier and with much higher virus load compared to recipients housed with vaccinated donor birds.
2. All species studied to date demonstrate a preference for certain codons over other synonymous codons (codon bias), a preference which is also observed for pairs of codons (di-codon bias). ARS researchers in East Lansing, Michigan, previously analyzed di-codon usage in the 18,742 referenced chicken genes and 86 protein-coding genes in the Md5 strain of Marek’s disease virus (MDV) and found a clear bias for preferential use of some di-codons and rare utilization of other di-codons. They replaced commonly used di-codons with synonymous uncommonly used di-codons for the MDV genes UL27 (glycoprotein B) and UL54 (ICP27), a transactivator of immediate early genes, which both led to reduced pathogenicity including a pronounced decrease in tumors and increased survivability compared to the control. To explain these findings, an assay was recently developed to measure changes in gene expression using a dual-promotor plasmid with self-cleaving fluorescent tag, which confirmed reduced gene expression of the deoptimized genes. These results demonstrate that altering the di-codon bias of select herpesvirus genes can affect the pathogenicity of the virus and may provide a novel method for MDV vaccine attenuation.
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 developed for protection against other common poultry viruses. These recombinant HVT vector (rHVT) vaccines have been shown to offer similar protection against Marek’s disease virus (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 in 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 were conducted using pairs of rHVT vaccines administered together followed by inoculation with Newcastle disease virus, infectious bursal disease virus, or infectious laryngotracheitis virus. A loss of protective efficacy was demonstrated against some viruses following certain vaccine combinations. 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. Although Marek’s disease (MD) vaccines have been in use for several decades, the exact mechanism of vaccine-induced protection is unknown. It is believed that the innate immune system plays a role in vaccine-induced immunity against pathogenic strains of Marek’s disease virus (MDV). To shed light on the possible role of the innate immunity on vaccine-mediated protection, ARS researchers in East Lansing, Michigan, investigated the effect of vaccination, Rispens/CVI988 and Herpes virus Turkey (HVT), on the activation of Natural killer (NK) cells by analyzing the expression pattern of CD107a, an activation marker for NK cells, using flow cytometry. The expression of CD107a was marginally upregulated by day second post vaccination and was significantly higher on days 3 and 4 post vaccination when compared to the expression levels of CD107a in the NK cells of age-matched control birds. HVT-induced activation of NK cells was only observed at day 4 post inoculation. This study shows for the first time that cellular components of innate immune system play a critical role in vaccine-induced protection. Adoptive transfer of lymphocytes from vaccinated birds with and without NK cells will provide further insights into the specific role of NK cells in vaccine-mediated protection.
5. A comprehensive gene expression profiling within the skin of Marek’s disease virus (MDV)-infected chickens of a highly-inbred Marek’s disease (MD)-susceptible chicken line. Profiling was conducted via next generation RNA sequencing at 10, 20, and 30 dpi by ARS researchers in East Lansing, Michigan. More than 20 MDV genes were significantly up regulated in the infected skin tissues at 20 and 30 dpi when compared to 10 dpi. A Real-Time PCR-based comparative gene expression analysis between skin and spleen of the same MDV-infected chickens revealed substantial differences in viral gene expression pattern between the two tissues. Very few viral genes were up regulated in the infected spleens when compared to the skin tissues at 10, 20, or 30 dpi. It is apparent that during the latent stage of infection, unlike the visceral tissues with very little viral activities, MDV is actively replicating within the Feather follicle epithelium (FFE) cells and expressing genes that are critical to the infection and dissemination of MDV. Identification and characterization of these genes with high transcriptional activities in the skin of virus-infected chickens could lead to the development of new recombinant vaccines to block the replication and shedding of MDV. This study will be the base for the development of specific recombinant vaccine to block the production and dissemination of such virus particles into the environment.
6. rMd5delta-meq is a recombinant vaccine that protects chickens against highly pathogenic Marek’s disease virus (MDV) strains better than all the commercially existing vaccines. The vaccine, however, causes bursal/thymic atrophy (BTA) in antibody negative chickens. To identify MDV gene(s) associated with BTA and alleviate the drawback, ARS researchers in East Lansing, Michigan, have generated recombinant constructs with single and double deletions of viral telomerase that might be used as effective and safe vaccines.
7. Traditional vaccines against infectious laryngotracheitis although protective, can revert to virulence and infect naïve flocks. This genetic instability is further exacerbated by the tendency of recombination between infectious laryngotracheitis virus (ILTV) strains. To generate a stable molecular clone, ARS scientists in Athens, Georgia, characterized a series of overlapping cosmid and yeast centromere plasmid clones that, when transfected into Longhorn male hepatoma cells, reconstituted viable virus. Studies in cell culture demonstrate the reconstituted virus grew to similar titers as those of the parental virulent strain. More importantly, in vivo pathogenicity studies conducted in collaboration with scientists at the University of Georgia demonstrated that chickens infected with the reconstituted virus exhibited clinical signs that were not statistically difference from those clinical sign scores of bird infected with the virulent USDA challenge strain. This suggests the reconstituted virus retained the genetic characteristics necessary for virulence. Often infectious clones of viruses are attenuated due to mutations that were inadvertently introduced during the cloning process. These ILTV clones which can be easily manipulated in vitro, will be used to generate stable molecularly defined vaccine strain.
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