2008 Annual Report 1a.Objectives (from AD-416) • Determine the role of avian astroviruses, avian reoviruses, avian rotaviruses and novel enteric viruses in inducing disease and production losses in poultry. • Characterize the molecular, physico-chemical, and antigenic properties of viruses that induce enteric disease and production losses in poultry. • Develop practical control strategies and diagnostic techniques for viruses causing enteric disease and production losses. 1b.Approach (from AD-416) Avian astroviruses, avian reoviruses and avian rotaviruses, among others, have been identified as the predominant virus families in specimens from commercial poultry flocks presenting with enteric disease. Initial work with each virus family shows a high level of genetic variation among isolates suggesting that:. 1)disease induction is strain dependent, and. 2)multiple serotypes of each virus are present in the field. Therefore, a minimum of 2 to 3 genetically variant isolates from enteric disease cases from each virus family will be evaluated for their pathogenesis. Pathogenesis studies will evaluate clinical, gross and microscopic lesions after exposure. Body weights will be used as a primary metric of disease severity. Virus shed times, immune dysfunction indicators and tissue tropism will also be evaluated. Once a virus is determined to be a cause of enteric disease as either a primary or pre-disposing agent, the isolate will be characterized further. Such characterization will include the development of specific antibody directed to the isolate and subsequent antigenic characterization by cross-neutralization assay. In depth genetic information will be collected and analyzed from target viruses by sequencing the entire genome and comparing with previously reported gene sequences from viruses of the same families in poultry and other species. With this information diagnostic tests that target virulent strains will be developed and bench validated. Two diagnostic test formats will be utilized, PCR based methods and micro-array based methods. Further characterization of the viruses will include evaluating: environmental stability, transmission characteristics, including age-related susceptibility to infection and disease. 3.Progress Report In cooperation with industry stakeholders and university collaborators, continued poultry enteric virus survey work using molecular diagnostic tools developed at Southeast Poultry Research Laboratory (SEPRL). This included a project in collaboration with researchers at North Carolina State University to determine the effect of management techniques on the enteric virus status in turkey flocks, a project with an industry stakeholder to determine the prevalence of avian astrovirus, rotavirus and reovirus in several commercial turkey flocks over time, and a survey of the enteric virus prevalence in commercial turkey flocks in the Midwestern United States. This research directly addresses our desire to identify and characterize the pathogens responsible for poultry enteric disease as outlined in National Program 103 Animal Health Action Plan, Component 6 Prevention and Control of Enteric Diseases, Problem Statement 6B Enteric Diseases of Poultry. Using the mammalian cell culture, applied and modified a technique for the isolation of avian rotaviruses directly from poultry intestinal field samples. This protocol isolates turkey-origin rotaviruses in large amounts, facilitating molecular characterization and sequencing efforts, and effectively removes other viruses usually present in field samples, namely avian reovirus and astrovirus. Pure rotavirus stocks are essential for downstream work on pathogenesis in experimental birds, and will allow the further characterization of the pathogens responsible for poultry enteric disease. This addresses our need to identify and characterize pathogens responsible for poultry enteric disease complexes as outlined in National Program 103 Animal Health Action Plan, Component 6 Prevention and Control of Enteric Diseases, Problem Statement 6B Enteric Diseases of Poultry. Continued nucleic acid sequencing of genes and genome segments of interest in isolated avian reoviruses, rotaviruses and astroviruses of turkey and chicken origin. Specifically, sequencing efforts concentrated on novel avian astroviruses from turkeys and chickens, selected genes from recent turkey rotavirus isolates, and selected genes and genome segments of turkey- and chicken-origin reoviruses. These efforts will contribute to Southeast Poultry Research Laboratory's pathogen-specific enteric virus diagnostic efforts. Performed pathogenesis work in commercial turkeys with isolates of astrovirus, rotavirus and reovirus, both alone and in combination (concomitant infections). This pathogenesis work was designed to replicate conditions in the field, where infections with multiple enteric viruses are common. Pathogenesis work is an essential step in the characterization of pathogens associated with poultry enteric disease. This addresses our need to identify and develop pathogen-specific markers useful for molecular or immunological detection of enteric pathogens as outlined in National Program 103 Animal Health Action Plan, Component 6 Prevention and Control of Enteric Diseases, Problem Statement 6B Enteric Diseases of Poultry. 4.Accomplishments 1. Completion of national and regional surveys of commercial poultry for the presence of enteric viruses. Up to date information on the prevalence of enteric viruses in commercial chickens and turkeys is essential for the control of enteric disease. A longitudinal survey of North Carolina commercial turkey farms and a national survey of commercial turkey and chicken operations were undertaken with stakeholder cooperation to determine the prevalence of several important enteric viruses. The data collected will inform the poultry industry and poultry researchers of the status of enteric disease in poultry operations in the United States (U.S.), and has provided the genetic data necessary to update molecular diagnostic tests for poultry enteric viruses. This accomplishment addresses our need to develop molecular tools to study the epidemiology and ecology of enteric pathogens as outlined in National Program 103 Animal Health Action Plan, Component 6 Prevention and Control of Enteric Diseases, Problem Statement 6B Enteric Diseases of Poultry. 2. Development of multiplex real time revere transcriptase polymerase chain reaction(RT-PCR) assay for the differential detection of turkey astrovirus types 1 and 2, chicken astrovirus, avian nephritis virus, and avian rotavirus. Poultry populations, flocks, and individual birds often have mixed infections with several enteric viruses. Recent enteric virus surveys have revealed several strains of avian astrovirus circulating in turkey and chicken populations in the United States. To improve molecular diagnostic tests for enteric viruses, a multiplex real time RT-PCR assay was developed and validated for the simultaneous detection of turkey astrovirus type-1, turkey astrovirus type-2, avian nephritis virus, and avian rotavirus in turkeys, and of chicken astrovirus, avian nephritis virus, and avian rotavirus in chickens. This test will streamline the testing of poultry flocks for the common viruses associated with enteric disease. This accomplishment addresses our need to identify and characterize pathogens responsible for poultry enteric disease complexes as outlined in National Program 103 Animal Health Action Plan, Component 6 Prevention and Control of Enteric Diseases, Problem Statement 6B Enteric Diseases of Poultry. 3. Molecular characterization of the turkey origin reovirus small genome segment 1(S1) genome segment. Several co-circulating strains of avian reovirus have recently been described in commercial turkeys in the U.S. In order to better characterize these novel viruses at the molecular level, the gene that encodes the turkey-origin reovirus cell-attachment protein sigmaC was sequenced and compared to existing sequences in national databases. The analysis revealed that the turkey-origin reoviruses are unique among the known avian reoviruses, and revealed unique properties of the sigmaC protein that may be involved in host specificity and enteric disease severity. This sequence data and analysis will allow the development of specific molecular diagnostic tests and has revealed proteins that may be useful in the development of immunological-based detection methods. This accomplishment addresses our need to identify and develop pathogen-specific markers useful for molecular or immunological detection of enteric pathogens as outlined in National Program 103 Animal Health Action Plan, Component 6 Prevention and Control of Enteric Diseases, Problem Statement 6B Enteric Diseases of Poultry. 4. Detection and molecular characterization of parvoviruses associated with poultry enteric disease. Although many avian viruses are associated with the poultry intestinal tract, their roles in enteric disease and poultry performance are not fully understood. In order to determine if uncharacterized viruses may play a role in poultry enteric disease syndromes, a molecular screening method was used to detect unknown viruses in chickens and turkeys exhibiting enteric disease signs. This approach revealed the presence of parvovirus deoxyribonucleic acid (DNA) in clinical samples, and allowed the sequence analysis of a substantial portion of the parvovirus genome. The identification of novel viruses that may play a significant role in poultry enteric disease is an important step toward an understanding of the etiology of these syndromes. This accomplishment addresses our need to identify and characterize pathogens responsible for poultry enteric disease complexes as outlined in National Program 103 Animal Health Action Plan, Component 6 Prevention and Control of Enteric Diseases, Problem Statement 6B Enteric Diseases of Poultry. 5.Significant Activities that Support Special Target Populations None. 6.Technology Transfer Review Publications Spackman, E., Pantin Jackwood, M.J., Day, J.M., Jackwood, M.W. 2008. Enteric viruses detected by molecular methods in commercial chicken and turkey flocks in the United States between 2005 and 2006. Avian Diseases. 52:235-244. Pantin Jackwood, M.J., Spackman, E., Day, J.M. 2008. Pathogenesis of type 2 turkey astrovirus with variant capsid genes in two-day-old poults. Avian Pathology. 37:193-201. Day, J.M., Pantin Jackwood, M.J., Spackman, E. 2007. A multiplex RT-PCR test for the differential identification of turkey astrovirus type-1, turkey astrovirus type-2, chicken astrovirus, avian nephritis virus and avian rotavirus. Avian Diseases. 51:681-684. Pantin Jackwood, M.J., Spackman, E., Day, J.M., Rives, D. 2007. Periodic monitoring of commercial turkeys for enteric viruses indicates continuous presence of astrovirus and rotavirus on farms. Avian Diseases. 51:674-680. Day, J.M., Pantin Jackwood, M.J., Spackman, E. 2007. Sequence and phylogenetic analysis of the S1 Genome segment of turkey-origin reoviruses. Virus Genes. 35:235-242. Zsak, L., Strother, K.O., Kisary, J. 2008. Partial genome sequence analysis of parvoviruses associated with enteric disease of poultry. Avian Pathology. 37:435-441.