Location: Endemic Poultry Viral Diseases Research2016 Annual Report
1a. Objectives (from AD-416):
1. Determine the virus microbiome of poultry from different geographical regions in the United States and complete the full genome sequence of poultry enteric viruses using metagenomics approaches. 2. Develop molecular diagnostic tests to determine the prevalence of novel poultry enteric viruses on poultry farms and their geographical distribution. 3. Identify host and/or viral genetic determinants that control pathogenicity, transmission, and drive the evolution of novel poultry enteric viruses. 4. Develop molecular vaccine platforms that will lead to highly efficacious vaccines that have been rationally designed to control enteric diseases of poultry, including mass delivery capability and companion diagnostics to differentiate naturally infected from vaccinated birds.
1b. Approach (from AD-416):
There is a pressing need to identify the novel viruses present in the poultry gut—an important first step in determining their role(s) in enteric disease and production losses. Recent efforts in our laboratory using the next generation of nucleic acid sequencing and related techniques to discover and characterize novel viruses in the poultry gut have been very successful, and suggest that our knowledge of the poultry gut virus community is incomplete. It is possible that unidentified viruses or viral communities may play specific roles in enteric disease syndromes and can act as predictors of enteric disease. Therefore, the use of high-throughput, sequence-independent pyrosequencing technology (next generation sequencing and associated technologies) and metagenomic analysis techniques will allow the discovery and characterization of novel RNA and DNA poultry enteric viruses and viral communities, allowing the association of certain etiologic agents with the poultry enteric disease syndromes noted in the field. Proper and effective management of poultry enteric disease will require novel, up-to-date diagnostic assays in order to determine the prevalence of enteric viruses on farms and to characterize the pathology caused by enteric viral infection. Based upon the full genome and/or gene sequences of novel poultry enteric viruses discovered and initially characterized using high-throughput pyrosequencing and metagenomic analyses, conserved sequences will be identified as targets for molecular diagnostic tests. These tests will be designed and validated in our laboratory and made available to industry partners and the research community at large. Finally, a successful control strategy for poultry enteric disease must include novel vaccine platforms that have been specifically designed to improve flock performance, lessen disease severity, and reduce viral transmission in the field. A targeted approach will be used to design recombinant (viral) vaccines using live turkey/chicken enterotropic viruses as expression vectors for specific enteric virus proteins deemed to be disease-associated using bioinformatic approaches to analyze enteric viral nucleic acid.
3. Progress Report:
5 Year Summary of the Project. Our recent and ongoing community-based analyses of the viruses present in the poultry gut have revealed numerous novel enteric viruses. The characterization of these novel viruses has been a major focus of this project, along with investigations of novel vaccine platforms to target the poultry enteric viruses. Our approach has involved the design and validation of molecular diagnostic assays for novel enteric viruses, and the use of these assays to survey archived and field samples for enteric viruses such as picobirnavirus, picornavirus, parvovirus, and coronavirus. An in-house bioinformatic analysis pipeline was devised to ensure reproducible, consistent handling of enteric samples that are selected for metagenomic (community-based) analysis. Project scientists developed a recombinant vaccine vector using a Newcastle disease virus (NDV) vaccine strain that specifically targets poultry intestinal tissue. Constructs containing enteric viral proteins, such as chicken parvovirus VP2 gene and turkey coronavirus (TCoV) S gene, have been constructed. These vaccines can be used to express foreign genes from multiple viruses implicated in poultry enteric disease. Further, project scientists evaluated NDV LaSota vaccine strain-based recombinant viruses expressing the glycoproteins B (gB) or D (gD) of infectious laryngotracheitis virus (ILTV) in broiler chickens in the presence of maternally-derived antibodies (MDA) against NDV. The results suggested that the NDV MDA moderately suppressed the immune response to the NDV vector. However, the ILTV antigens expressed from the NDV vector were still sufficient to confer a significant protection against ILT clinical disease in birds. Thermostable NDV vaccines have been used widely to control Newcastle disease (ND) in village flocks, since they do not rely on cold chains for delivery and storage. To explore the potential use of a thermostable NDV as a vaccine vector, NDV vaccine strain TS09-C vectoring a green fluorescent protein (GFP) gene was generated using reverse genetics technology. The recombinant virus retained the same thermostability as its parental virus. Vaccination of specific pathogen free (SPF) chickens conferred complete protection against virulent NDV. The results suggested that the rTS09-C virus could be used as a vaccine vector to develop thermostable vaccines against ND and avian enteric diseases in developing countries. In cooperation with industry stakeholders, a comparative analysis of SPF chickens placed on broiler farms with historic enteric disease revealed numerous suspect enteric viruses that may be involved in disease and performance problems. Analysis of the intestinal viral community in broiler chickens revealed a role for the poultry enteric picornaviruses in the enteric syndromes noted in the field. This investigation provided insight into the colonization of SPF chickens by intestinal microorganisms under field conditions and compared the pre-contact intestinal microbial communities (the gut “microbiome”) with the altered gut microbiome following contact with poultry raised in the field. Analysis of the intestinal virus community from contact birds (“sentinels”) revealed colonization of the gut by viruses that were not present in pre-contact birds or present in proportionally lower numbers. Analysis of the sentinel gut bacterial community revealed an altered community in the post-contact birds, notably by members of the Lachnospiracea/Clostridium and Lactobacillus groups of bacteria. Investigations of the intestinal viral community in the poultry gut have provided some insight into the geographical distribution and the taxonomy of the avian enteric picornaviruses. We completed a comparative analysis of the gut virome from a healthy turkey flock versus a flock placed in the field and experiencing signs of enteric disease. This investigation revealed a number of enteric picornavirus sequences that were present in the commercial birds in the field but that were absent in the healthy flock. A novel molecular diagnostic assay was designed and used to track the shedding of field strains of turkey enteric picornavirus in poults inoculated with picornavirus-positive intestinal homogenates prepared from turkeys that were experiencing moderate enteric disease. The propagation of this novel enteric picornavirus in poults resulted in significant reduction in weight gain, and the birds continually shed picornavirus in feces during the experiment; this suggests that this common inhabitant of the turkey gut may result in performance problems or enteric disease in the field. Pathogenesis experiments in broiler chickens using novel parvoviruses isolated from the intestines of chickens affected by enteric diseases were completed as well. Experimental infection of chickens resulted in characteristic signs of enteric disease, including signs consistent with runting-stunting syndrome (RSS), such as reduction in weight gain. Shedding of parvovirus in the feces was detected via a molecular diagnostic assay, and the subsequent presence of virus in the blood was observed. Parvovirus could be detected in the intestines of inoculated birds at 7 and 14 days following inoculation, and in most of the major organs for weeks after inoculation. These data indicate that chicken parvovirus alone can induce signs consistent with RSS in broilers and may be an important determinant in the complex etiology of the enteric disease syndromes. Further analysis revealed a strong genetic similarity between the chicken and turkey parvoviruses. We further characterized the evolutionary and biological relationships of poultry parvoviruses with the use of comparative full-length genome sequence analyses and studied host-specific virus replication after infection of day-old chickens and turkeys. Both the chicken and turkey parvoviruses are highly adapted to their respective hosts, and the viral VP1 gene is primarily responsible for this host specificity. A fully validated molecular diagnostic assay targeting the novel turkey picobirnaviruses (PBVs) was also developed. The novel PBVs were first detected via metagenomic analyses of the turkey gut virome by project scientists, resulting in the first avian PBV sequences deposited in the public databases. Phylogenetic analysis comparing novel North Carolina turkey-origin PBV with genogroup I and II PBVs detected in several mammal species revealed the turkey-origin PBVs are unique among the available sequences. Interestingly, the turkey origin PBVs could be genotyped as genogroup I PBVs, but do not appear to group closely with representative members of the recognized genogroup I or II PBVs, suggesting the turkey PBVs have relatively recently diverged from a genogroup I PBV. An outbreak of turkey enteric coronavirus (TCoV) was investigated by project scientists, and the re-emerging TCoVs were characterized. In response to requests from the field numerous samples were characterized via an in-house diagnostic assay for TCoV. The results of the assay allowed industry stakeholders to make management decisions in a timely manner, and proved to be a good indicator of subsequent seroconversion by TCoV positive flocks. Project scientists further completed metagenomic analysis, sequencing, and phylogenetic analysis of these re-emerging TCoV isolates, demonstrating that they are unique when compared to past isolates of TCoV and their geographical distribution.
1. Molecular basis for the thermostability of Newcastle disease virus. Thermostable Newcastle disease virus (NDV) vaccines have been used widely to protect village chickens against Newcastle disease (ND) for decades. However, the genetic basis underlying the NDV thermostability is poorly understood. Project scientists generated chimeric viruses by exchanging viral genes between the thermostable TS09-C strain and thermolabile LaSota strain using reverse genetics technology. Evaluations of these chimeric NDVs demonstrated that the HN protein of NDV is a crucial determinant of thermostability, and the HN gene from a thermostable NDV could be engineered into a thermolabile NDV vaccine strain for developing a novel NDV vaccine which will improve vaccine thermostability and protection efficacy.
2. In this study all currently known chicken picornaviruses including a novel one (chicken phacovirus 1) were identified by viral metagenomic and RT-PCR methods from a single sample of a diarrheic chicken in Hungary suffering from an infection with a total of eight picornaviruses. Hungarian collaborators determined the complete genomes of six of the eight picornaviruses and analyzed them in detail, including genomic and phylogenetic analyses and secondary RNA structural modeling of 5’/3’ UTRs. The identified picornaviruses belong to genera Sicinivirus (first complete genome), Gallivirus, Tremovirus, Avisivirus, “Orivirus” (two potential genotypes) and “Phacovirus” which is a novel proposed genus. As a result, similar sequences were discovered in the online enteric metagenomic public databases deposited and analyzed by our laboratory at USDA/ARS/SEPRL. As a result, ARS scientist also detected the novel phacoviruses in multiple samples of chickens from USA (multiple regions in Arkansas). Phylogenetic analyses of these novel phacoviruses from turkeys and chickens continues.
3. Recent metagenomic analyses of the enteric viromes in turkeys and chickens by our laboratory have revealed complex viral communities comprised of multiple viral families. Of particular significance are the novel avian picobirnaviruses (Family Picobirnaviridae), multiple genera of tailed phages (Family Siphoviridae), and undescribed avian enteric picornaviruses (Family Picornaviridae). In addition to these largely undescribed—and therefore relatively poorly understood—poultry enteric viral families, these metagenomic analyses have also revealed the presence of well-known groups of enteric viruses such as the chicken and turkey astroviruses (Family Astroviridae) and the avian rotaviruses and reoviruses (Family Reoviridae; Subfamily Sedoreovirinae). The order Picornavirales is a group of viruses in flux, particularly among the avian picornaviruses, as several new genera have been described recently based upon community analysis of enteric viromes from poultry and other avian species worldwide. Our previous investigation of the turkey enteric picornaviruses suggests the avian enteric picornaviruses may contribute to the enteric disease syndromes and performance problems often observed in turkeys in the Southeastern United States. Our recent phylogenetic analysis of turkey and chicken enteric samples archived at the Southeast Poultry Research Laboratory from 2004 to present is a first step in placing these novel avian picornaviruses within the larger Picornaviridae Family.
5. Significant Activities that Support Special Target Populations:
Wen, G., Hu, X., Zhao, K., Wang, H., Zhang, Z., Zhang, T., Yang, J., Luo, Q., Zhang, R., Pan, Z., Shao, H., Yu, Q. 2016. Molecular basis for the thermostability of Newcastle disease virus. Scientific Reports. 6:22492. doi: 10.1038/srep22492.
Zsak, L., Cha, R., Day, J.M. 2013. Chicken parvovirus-induced runting-stunting syndrome in young broilers. Avian Diseases. 57(1):123-127. doi: 10.1637/10371-091212-ResNote.1.
Day, J.M., Zsak, L. 2016. Molecular characterization of enteric picornaviruses in archived turkey and chicken samples from the United States. Avian Diseases. 60(2):500-505. doi: 10.1637/11289-092415-ResNote.
Day, J.M., Oakley, B., Seal, B.S., Zsak, L. 2015. Comparative analysis of the intestinal bacterial and RNA viral communities from sentinel birds placed on selected broiler chicken farms. PLoS One. doi: 10.1371/journal.pone.0117210.
Schutta, C., Barrera, J., Pisano, M., Zsak, L., Grubman, M.J., Mayr, G.A., Moraes, M.P., Kamicker, B.J., Brake, D.A., Ettyreddy, D., Brough, D.E., Butman, B.T., Neilan, J.J. 2016. Multiple efficacy studies of an adenovirus-vectored foot-and-mouth disease virus serotype A24 subunit vaccine in cattle using direct homologous challenge. Vaccine. 34(27):3214-3220. doi: 10.1016/j.vaccine.2015.12.018.
Bai, F., Tian, H., Yu, Q., Ren, G., Li, D. 2015. Expressing foreign genes by Newcastle disease virus for cancer therapy. Molecular Biology. 49(2):171-178. DOI: 10.1134/S0026893315020028.
Zhao, W., Spatz, S.J., Zsak, L., Yu, Q. 2016. Generation of Newcastle diease virus (NDV) recombinants expressing the infectious laryngotracheitis virus (ILTV) glycoprotein gB or gD as dual vaccines. In: Thomas, S.,editor. Vaccine Design: Methods and Protocols. volume 2. New York, NY: Springer Science+Business Media. p.89-101.
Niu, Z., Bai, F., Sun, T., Tian, H., Yu, D., Yin, J., Li, S., Li, T., Cao, H., Yu, Q., Wu, Y., Ren, G., Li, D. 2014. Recombinant Newcastle disease virus expressing IL15 demonstrates promising antitumor efficiency in melanoma model. Technology in Cancer Research and Treatment. DOI: 10.7785/tcrt.2012.500414.
Wu, C., Day, J.M. 2016. Enteric viruses. In: Williams, S.M., Dufour-Zavala, L., Jackwood, M.W., Lee, M.D., Lupiani, B., Reed, W.M., Spackman, E., Woolcock, P.R., editors. Isolation, Identification and Characterization of Avian Pathogens. 6th edition. Madison, WI: OmniPress, Inc. p.199-214.
Boros, A., Pankovics, P., Adonyi, A., Fenyvesi, H., Day, J.M., Gia Phan, T., Delwart, E., Reuter, G. 2015. A diarrheic chicken simultaneously co-infected with multiple picornaviruses: Complete genome analysis of avian picornaviruses representing up to six genera. Virology. 489:63-74. doi: 10.1016/j.virol.2015.12.002.