Progress 11/17/06 to 09/30/11
Outputs
Progress Report Objectives (from AD-416) 1. Characterize the interaction of virus replication and macrophage responses. 2. Identify natural genetic variation associated with disease susceptibility. Approach (from AD-416) Identification of specific pathways that associate with variation in porcine reproductive and respiratory syndrome virus (PRRSV) replication and macrophage function leading to novel gene targets for the control of PRRSV infection. Alveolar macrophages will be obtained from diverse populations of swine and evaluated for their ability to support replication of PRRS viruses. Replication parameters will be estimated and macrophages that support either high or low levels of virus replication will be selected for studies of gene expression. Identifying PRRSV genotypes that confer fitness in macrophages, and host genes that respond to PRRSV fitness, to provide novel targets for intervention and control of PRRSV infections. These studies will use adapted isolates to identify viral genotypes that correlate with fitness of PRRSV in porcine alveolar macrophages and corresponding changes in macrophage transcriptional profiles. Genetic variation in specific ovine genes influences predisposition to ovine lentivirus (OLV) and the associated disease, ovine progressive pneumonia (OPP). We will thoroughly evaluate the most obvious candidate genomic regions for effects on lentiviral disease, like that containing CCR5. Our aim is to evaluate important regions of the genome for allelic association with the OLV disease susceptibility and progression phenotypes. Selection of regions will be based on a variety of scientific observations including, but not limited to, comparative mammalian biology. A selected set of 90 single nucleotide polymorphism (SNP) markers will be identified that are highly-informative in beef and dairy cattle. The development of this marker set represents non-hypothesis-driven research. The markers and genotyping assays for the markers will be readily available for any traceback needs. The same markers are also ideal for animal identification (i.e., sample matching) and routine parentage analysis. After ear tags and other physical identification devices have been removed, an animal�s DNA remains as a stable, accessible, integral, and identifiable component of its products and, thus, provides a gold standard for auditing the fidelity of physical labels and associated records. Identification of ovine progressive pneumonia virus (OPPV) genetic subtypes. Recently, we identified key mutations within an ovine gene that confer reduced OPPV susceptibility. To determine whether the reduced susceptibility is specific for OPPV strains, and whether OPPV genetic subtypes may potentially influence host susceptibility to infection and/or disease, we developed an OPPV phylotyping system. Both the group specific antigen (gag) and the envelope (env) genes are used to phylotype OPPV genetic subtypes. We are currently identifying OPPV genetic subtypes in sheep with or without the key mutations that reduce OPPV susceptibility. This will allow us to test for viral strains that may influence host susceptibility to OPP. Informatics for studying the transcriptome of swine infected with PRRSV. In April 2011, the Swine Genome Sequencing Consortium released Build 10 of the swine genome (Ssc10) with much improved transcript annotation (in quality and scope) over that which was available in 2010. During 2010, we analyzed the data generated by scientists at the USDA, ARS, National Animal Disease Center, Ames, IA, to investigate genes involved in the progression of PRSSV, pseudorabies virus (PRV), swine influenza virus (SIV), and porcine circovirus type 2 (PCV2) in infected swine as part of a National Pork Board grant (effective 11/01/2008 to 11/01/2010, entitled �Gene expression in lymph nodes of PRRSV-infected pigs,� Accession Number: 415042, Project Number: 3625-32000-088-28R). Next-generation sequencing was used to create libraries of 10 million �SAGE-like� 20-bp tags at days 1, 3, 7 and 14 post-infection in both infected and control groups of swine. These data were reanalyzed using Ssc10 as the reference, and have provided evidence of many more unambiguous tag to transcript to gene to pathway associations than what was possible in 2010. Accomplishments 01 Discovery of a gene for pneumonia susceptibility in sheep. Ovine progressive pneumonia is an incurable slow-acting disease of sheep cause by persistent lentivirus infection. The Animal and Plant Health Inspecti Service (APHIS) has estimated that 36% of U.S. sheep operations have infected animals. Infected ewes have fewer lambs, raise lighter litters, and need to be replaced sooner than uninfected ewes. ARS scientists identified a major gene conferring susceptibility to ovine progressive pneumonia. The ancestral protein encoded by this gene is highly susceptible to the virus, but naturally occurring mutations in this gene have resulted in decreased susceptibility. Sheep without any natural functional copies of this gene appear to be highly resistant to infectio and live long, healthy, productive lives. ARS researchers at Clay Center NE; Pullman, WA; and Dubois, ID, verified the effect of this gene in multiple flocks. This discovery paves the way for breeding sheep that a significantly less likely to become infected.
Impacts
(N/A)
Publications
- Harhay, G.P., Smith, T.P.L., Alexander, L.J., Haudenschild, C.D., Keele, J. W., Matukumalli, L.K., Schroeder, S.G., Van Tassell, C.P., Gresham, C.R., Bridges, S.M., Burgess, S.C., Sonstegard, T.S. 2010. An atlas of bovine gene expression reveals novel distinctive tissue characteristics and evidence for improving genome annotation. Genome Biology [online serial]. 11:R102.
- Miller, L.C., Neill, J.D., Harhay, G.P., Lager, K.M., Laegreid, W.W., Kehrli, Jr., M.E. 2010. In-depth global analysis of transcript abundance levels in porcine alveolar macrophages following infection with porcine reproductive and respiratory syndrome virus. Advances in Virology. 2010:Article 864181. Available:
- Murdoch, B.M., Clawson, M.L., Yue, S., Basu, U., McKay, S., Settles, M., Capoferri, R., Laegreid, W.W., Williams, J.L., Moore, S.S. 2010. PRNP haplotype associated with classical BSE incidence in European Holstein cattle. PLoS One [serial online]. 5(9): e12786.
- Durso, L.M., Harhay, G.P., Bono, J.L., Smith, T.P. 2011. Virulence- associated and antibiotic resistance genes of microbial populations in cattle feces analyzed using a metagenomic approach. Journal of Microbiological Methods. 84: 278-282.
- Durso, L.M., Harhay, G.P., Smith, T.P., Bono, J.L., Desantis, T.Z., Clawson, M.L. 2011. Bacterial community analysis of beef cattle feedlots reveals that pen surface is distinct from feces. Foodborne Pathogens and Disease. 8(5):647-649. Available: DOI: 10.1089/fpd.2010.0774.
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Progress 10/01/09 to 09/30/10
Outputs
Progress Report Objectives (from AD-416) 1. Characterize the interaction of virus replication and macrophage responses. 2. Identify natural genetic variation associated with disease susceptibility. Approach (from AD-416) Identification of specific pathways that associate with variation in porcine reproductive and respiratory syndrome virus (PRRSV) replication and macrophage function leading to novel gene targets for the control of PRRSV infection. Alveolar macrophages will be obtained from diverse populations of swine and evaluated for their ability to support replication of PRRS viruses. Replication parameters will be estimated and macrophages that support either high or low levels of virus replication will be selected for studies of gene expression. Identifying PRRSV genotypes that confer fitness in macrophages, and host genes that respond to PRRSV fitness, to provide novel targets for intervention and control of PRRSV infections. These studies will use adapted isolates to identify viral genotypes that correlate with fitness of PRRSV in porcine alveolar macrophages and corresponding changes in macrophage transcriptional profiles. Genetic variation in specific ovine genes influences predisposition to ovine lentivirus (OLV) and the associated disease, ovine progressive pneumonia (OPP). We will thoroughly evaluate the most obvious candidate genomic regions for effects on lentiviral disease, like that containing CCR5. Our aim is to evaluate important regions of the genome for allelic association with the OLV disease susceptibility and progression phenotypes. Selection of regions will be based on a variety of scientific observations including, but not limited to, comparative mammalian biology. A selected set of 90 single nucleotide polymorphism (SNP) markers will be identified that are highly-informative in beef and dairy cattle. The development of this marker set represents non-hypothesis-driven research. The markers and genotyping assays for the markers will be readily available for any traceback needs. The same markers are also ideal for animal identification (i.e., sample matching) and routine parentage analysis. After ear tags and other physical identification devices have been removed, an animal�s DNA remains as a stable, accessible, integral, and identifiable component of its products and, thus, provides a gold standard for auditing the fidelity of physical labels and associated records. 1) Genetic predisposition to ovine progressive pneumonia virus (OPPV) - A genome-wide association study of OPPV in sheep has revealed specific gene sequences that may predispose sheep to infection under some production environments. OPP research from four perspectives has been conducted: i) Host genetics - We have typed 54,241 SNPs on a set of case- control sheep and identified a gene and haplotypes associated with OPPV resistance/susceptibility. The expressed mRNA haplotypes of this gene have also been sequenced. ii) Pathogen genetics - PCR-based assays have been developed for detecting infected sheep by measuring OPPV proviral DNA integrated in the host genome and for characterizing OPPV strain variation. iii) Host serology - More than 4,500 U.S. Meat Animal Research Center (USMARC) sheep have been phenotyped for OPP using a competitive ELISA for OPPV serological status. iv) Bioinformatics - We have sequenced, assembled, and annotated the complete ovine gene that appears to influence OPP infection in our sheep (78 kb total, from four BACs, 170- fold coverage). The sequence and information have been deposited in GenBank for concurrent release with manuscript publication. 2) Informatics for studying the transcriptome of swine infected with PRRSV - Collaborated with National Animal Disease Center (NADC) to investigate genes involved in the progression of PRSSV, PRV, SIV, and PCV2 in infected swine. Next-generation sequencing was used to create libraries of 10 million �SAGE-like� 20-bp tags at days 1, 3, 7 and 14 post-infection in both an infected and control group of swine. Data has been processed though our pipeline that has associated the tags with transcripts, genes, and pathways. 3) Effect of shade on heat-stress induced immune function changes in cattle - Bovine Respiratory Disease Complex (BRDC) is one of the most costly diseases affecting cattle. Although BRDC is composed of an assortment of bacterial and viral pathogens, conditions which place cattle under physiological stress can exacerbate the disease. We have initiated a study to determine if the presence or absence of shade alters immune parameters in cattle, in particular, during times of extreme heat. Cattle are currently being exposed to four different types/densities of shade, and blood values are being collected weekly during this eight-week study. 4) Evaluation of antibody titers to BRDC vaccination and association with respiratory disease - It may be possible to identify animals that are more susceptible to BRDC soon after their arrival at a feedlot by measuring their immune response to prophylactic vaccination. Whole blood and serum have been collected from the USMARC's �disease resistance� herd of cattle to determine complete blood counts and antibody titers to IBR, PI3, and BRSV using a single three-way assay. This ImmunoComb� assay will allow us to calculate the association between high and/or low antibody titers and illness recorded during the animals� feeding-out period in the feedlot. Accomplishments 01 Improvement of a nucleotide polymorphism-based typing system for Escherichia coli O157:H7. Cattle are a reservoir of the recently emerge human pathogen E. coli O157:H7 and harbor distinct subtypes that do not all associate with human disease. A set of 178 nucleotide polymorphisms was developed in 2009 by ARS scientists at Clay Center, NE, that showed great utility in classifying E. coli O157:H7 genetic subtypes of cattle and/or human origin. To achieve higher resolution with this system, the scientists developed and applied a set of 720 nucleotide polymorphisms t 420 E. coli O157:H7 strains. This set effectively tags E. coli O157:H7 genetic diversity associated with humans and cattle, and an evolutionary analysis of the diversity indicates that cattle harbor subtypes that hav evolved away from an association with human disease. Consequently, the nucleotide polymorphism set depicts E. coli O157:H7 genetic diversity an shows how strains are evolutionarily related with high resolution. These qualities define it as a preferential method for detecting and distinguishing E. coli O157:H7 genetic subtypes involved in epidemiological investigations.
Impacts
(N/A)
Publications
- Decker, J.E., Pires, J.C., Conant, G.C., McKay, S.D., Heaton, M.P., Chen, K., Cooper, A., Vilkki, J., Seabury, C.M., Caetano, A.R., Johnson, G.S., Brenneman, R.A., Hanotte, O., Eggert, L.S., Wiener, P., Kim, J.J., Kim, K. S., Sonstegard, T.S., Van Tassell, C.P., Neibergs, H.L., McEwan, J.C., Brauning, R., Coutinho, L.L., Babar, M.E., Wilson, G.A., McClure, M.C., Rolf, M.M., Kim, J., Schnabel, R.D., Taylor, J.F. 2009. Resolving the Evolution of Extant and Extinct Ruminants With High-Throughput Phylogenomics. Proceedings of the National Academy of Sciences. 106(44) :18644-18649.
- Daude, N., Wohlgemuth, S., Rogaeva, E., Farid, A.H., Heaton, M.P., Westaway, D. 2009. Frequent Missense and Insertion/Deletion Polymorphisms in the Ovine Shadoo Gene Parallel Species-Specific Variation in PrP. PLoS One [serial online]. 4(8):e6538.
- Bono, J.L. 2009. Genotyping Escherichia coli O157:H7 for Its Ability to Cause Disease in Humans. Current Protocols in Microbiology. 14:5A.3.1-5A. 3.10.
- Heaton, M.P., Leymaster, K.A., Kalbfleisch, T.S., Freking, B.A., Smith, T. P., Clawson, M.L., Laegreid, W.W. 2010. Ovine Reference Materials and Assays for Prion Genetic Testing. BioMed Central (BMC) Veterinary Research [serial online]. 6:23. Available: http://www.biomedcentral.com/1746- 6148/6/23.
- Durso, L.M., Harhay, G.P., Smith, T.P.L., Bono, J.L., Desantis, T.Z., Harhay, D.M., Andersen, G.L., Keen, J.E., Laegreid, W.W., Clawson, M.L. 2010. Animal-to-Animal Variation in Fecal Microbial Diversity among Beef Cattle. Applied and Environmental Microbiology. 76(14):4858-4862.
- Donthu, R., Larkin, D.M., Heaton, M.P., Lewin, H.A. 2010. Discovery, Validation and Characterization of 1039 Cattle Single Nucleotide Polymorphisms. Animal Genetics. 41(4):421-423.
- Keen, J.E., Laegreid, W.W., Chitko Mckown, C.G., Durso, L.M., Bono, J.L. 2010. Distribution of Shiga-Toxigenic Escherichia coli O157 in the Gastrointestinal Tract of Naturally O157-Shedding Cattle at Necropsy. Applied and Environmental Microbiology. 76(15):5278-5281.
- Chitko Mckown, C.G., Macneil, M.D. 2010. Development of Fibroblast Cell Lines From the Cow Used to Sequence the Bovine Genome. Animal Genetics. 41(4):445.
- Murdoch, B.M., Clawson, M.L., Laegreid, W.W., Stothard, P., Settles, M., McKay, S., Prasad, A., Wang, Z., Moore, S.S., Williams, J.L. 2010. A 2cM Genome-Wide Scan of European Holstein Cattle Affected by Classical BSE. BioMed Central (BMC) Genetics [serial online]. 11:20. Available: http://www.biomedcentral.com/1471-2156/11/20.
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Progress 10/01/08 to 09/30/09
Outputs
Progress Report Objectives (from AD-416) 1. Characterize the interaction of virus replication and macrophage responses. 2. Identify natural genetic variation associated with disease susceptibility. Approach (from AD-416) Identification of specific pathways that associate with variation in porcine reproductive and respiratory syndrome virus (PRRSV) replication and macrophage function leading to novel gene targets for the control of PRRSV infection. Alveolar macrophages will be obtained from diverse populations of swine and evaluated for their ability to support replication of PRRS viruses. Replication parameters will be estimated and macrophages that support either high or low levels of virus replication will be selected for studies of gene expression. Identifying PRRSV genotypes that confer fitness in macrophages, and host genes that respond to PRRSV fitness, to provide novel targets for intervention and control of PRRSV infections. These studies will use adapted isolates to identify viral genotypes that correlate with fitness of PRRSV in porcine alveolar macrophages and corresponding changes in macrophage transcriptional profiles. Genetic variation in specific ovine genes influences predisposition to ovine lentivirus (OLV) and the associated disease, ovine progressive pneumonia (OPP). We will thoroughly evaluate the most obvious candidate genomic regions for effects on lentiviral disease, like that containing CCR5. Our aim is to evaluate important regions of the genome for allelic association with the OLV disease susceptibility and progression phenotypes. Selection of regions will be based on a variety of scientific observations including, but not limited to, comparative mammalian biology. A selected set of 90 single nucleotide polymorphism (SNP) markers will be identified that are highly-informative in beef and dairy cattle. The development of this marker set represents non-hypothesis-driven research. The markers and genotyping assays for the markers will be readily available for any traceback needs. The same markers are also ideal for animal identification (i.e., sample matching) and routine parentage analysis. After ear tags and other physical identification devices have been removed, an animal�s DNA remains as a stable, accessible, integral, and identifiable component of its products and, thus, provides a gold standard for auditing the fidelity of physical labels and associated records. Significant Activities that Support Special Target Populations Bovine Respiratory Disease Complex (BRDC) accounts for 75% of morbidity and 50% of mortality in feedlots. Its cost is cumulative and includes: treatment, lost production, as well as death. These losses make BRDC one of the most costly diseases affecting cattle. BRDC is composed of an assortment of bacterial and viral pathogens. One of these bacteria, Mannheimia haemolytica (formerly Pasteurella haemolytica), causes pneumonia in ruminants and produces a leukotoxin that is cytotoxic only for ruminant leukocytes (white blood cells). When viral and bacterial agents break down the antimicrobial barrier in the lungs, M. haemolytica--which is usually a commensal organism--can become pathogenic. In collaboration with scientists in the Department of Veterinary Biosciences at the University of Nebraska � Lincoln, we have initiated in vitro studies using bovine macrophages infected with M. haemolytica to determine if we can alter the levels of leukotoxin produced under different cell-culture conditions. We have determined that succinate is an important metabolite in the growth of M. haemolytica that can be used in place of supplemental serum in macrophage/bacterial cultures. We plan to exploit this finding in future metabolic studies of this important bovine pathogen. In collaboration with scientists in the Department of Homeland Security (DHS), U. S. Meat Animal Research Center (USMARC) scientists have been testing bovine, ovine, and porcine cell lines developed at USMARC for their suitability to support protein expression of a Foot and Mouth Disease (FMD) molecular vaccine. Four cell lines (MARC-BGCF2, MARC-BOE, Cdelta 2+, and Cdelta 2-) were screened by electron microscopy (EM) for their ability to support FMD protein expression; unfortunately, none of the cell lines supported detectable protein expression. These same four cell lines were screened using EM and virus isolation for their ability to support FMD virus (FMDV) expression. Cells were infected with two different FMDV strains. One of the cell lines, MARC-BOE, supported excellent replication of one of the FMDV isolates. Virus titers were similar to that achieved in the gold-standard cell line currently used for FMDV propagation. Development of informatics for studying the transcriptome of swine infected by porcine reproductive and respiratory syndrome virus (PRRSV). Collaborated with scientists at the National Animal Disease Center (NADC) to investigate the genes involved in the progression of PRSSV in infected swine. Scientists at NADC and USMARC were awarded a National Pork Board grant (effective 11/01/2008 to 11/01/2010) entitled �Gene expression in lymph nodes of PRRSV-infected pigs,� CRIS Project: 3625- 32000-088-28R. Over the past year, NADC and USMARC scientists have collaborated to coordinate the collection of data generated from this grant at NADC with its analysis at USMARC. NADC and USMARC scientists have adapted pre-existing data processing pipelines to analyze transcriptomic data from this project to gain new insights into the molecular mechanisms involved in PRRSV infection.
Impacts
(N/A)
Publications
- Kijas, J.W., Townley, D., Dalrymple, B.P., Heaton, M.P., Maddox, J.F., McGrath, A., Wilson, P., Ingersoll, R.G., McCulloch, R., McWilliam, S., Tang, D., McEwan, J., Cockett, N., Oddy, V.H., Nicholas, F.W., Raadsma, H. 2009. A Genome Wide Survey of SNP Variation Reveals the Genetic Structure of Sheep Breeds. PLoS One [serial online]. 4(3):e4668. Available: http://www.plosone.org.
- Elsik, C.G., Gibbs, R., Skow, L., Tellam, R., Weinstock, G., Worley, K., Kappes, S.M., Green, R.D., Alexander, L.J., Bennett, G.L., Carroll, J.A., Chitko Mckown, C.G., Hamernik, D.L., Harhay, G.P., Keele, J.W., Liu, G., Macneil, M.D., Matukumalli, L.K., Rijnkels, M., Roberts, A.J., Smith, T.P., Snelling, W.M., Stone, R.T., Waterman, R.C., White, S.N. 2009. The Genome Sequence of Taurine Cattle: A Window to Ruminant Biology and Evolution. Science. 324:522-528.
- Matukumalli, L.K., Lawley, C.T., Schnabel, R.D., Taylor, J.F., Allan, M.F., Heaton, M.P., O'Connell, J., Moore, S.S., Smith, T.P., Sonstegard, T.S., Van Tassell, C.P. 2009. Development and Characterization of a High Density SNP Genotyping Assay for Cattle. PLoS One. 4(4):e5350. Available: http://dx.doi.org/10.1371/journal.pone.0005350.
- Clawson, M.L., Keen, J.E., Smith, T.P., Durso, L.M., Mcdaneld, T.G., Mandrell, R.E., Davis, M.A., Bono, J.L. 2009. Phylogenetic Classification of Escherichia coli O157:H7 Strains of Human and Bovine Origin Using a Novel Set of Nucleotide Polymorphisms. Genome Biology [serial online]. 10:R56. Available: http://genomebiology.com/2009/10/5/R56.
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Progress 10/01/07 to 09/30/08
Outputs
Progress Report Objectives (from AD-416) 1. Characterize the interaction of virus replication and macrophage responses. 2. Identify natural genetic variation associated with disease susceptibility. Approach (from AD-416) Identification of specific pathways that associate with variation in porcine reproductive and respiratory syndrome virus (PRRSV) replication and macrophage function leading to novel gene targets for the control of PRRSV infection. Alveolar macrophages will be obtained from diverse populations of swine and evaluated for their ability to support replication of PRRS viruses. Replication parameters will be estimated and macrophages that support either high or low levels of virus replication will be selected for studies of gene expression. Identifying PRRSV genotypes that confer fitness in macrophages, and host genes that respond to PRRSV fitness, to provide novel targets for intervention and control of PRRSV infections. These studies will use adapted isolates to identify viral genotypes that correlate with fitness of PRRSV in porcine alveolar macrophages and corresponding changes in macrophage transcriptional profiles. Genetic variation in specific ovine genes influences predisposition to ovine lentivirus (OLV) and the associated disease, ovine progressive pneumonia (OPP). We will thoroughly evaluate the most obvious candidate genomic regions for effects on lentiviral disease, like that containing CCR5. Our aim is to evaluate important regions of the genome for allelic association with the OLV disease susceptibility and progression phenotypes. Selection of regions will be based on a variety of scientific observations including, but not limited to, comparative mammalian biology. A selected set of 90 single nucleotide polymorphism (SNP) markers will be identified that are highly-informative in beef and dairy cattle. The development of this marker set represents non-hypothesis-driven research. The markers and genotyping assays for the markers will be readily available for any traceback needs. The same markers are also ideal for animal identification (i.e., sample matching) and routine parentage analysis. After ear tags and other physical identification devices have been removed, an animal�s DNA remains as a stable, accessible, integral, and identifiable component of its products and, thus, provides a gold standard for auditing the fidelity of physical labels and associated records. Significant Activities that Support Special Target Populations Two porcine monocyte-derived cell lines, designated Cdelta2+ and Cdelta2-, were developed and characterized to determine their usefulness in studies of disease susceptibility. Since peripheral blood monocytes and their tissue counterparts, the macrophages, play an important role as the first immune response responders to infectious disease-causing pathogens, these cell lines will be an additional tool with which to study porcine pathogens in vitro. Analysis of PRRSV-infected swine macrophages. Changes in gene product abundance in PRRSV-infected swine macrophage cell cultures were analyzed as a function of infection time. The experimental serial analysis of gene expression (SAGE) data acquired in FY2007 was used in these analyses. These analyses associated the SAGE data with porcine and PRRSV mRNA sequences, genes, and biological pathways. Changes in gene product abundance were analyzed for statistical significance using multiple tests. National Program 103 - Animal Health; National Program Components - Problem Statements 2C: Genetic and Biological Determinants of Disease Susceptibility - Mucosal Diseases of Livestock and Poultry and 4B: Countermeasures to Prevent and Control Respiratory Diseases - Porcine Respiratory Diseases
Impacts
(N/A)
Publications
- Chitko Mckown, C.G., Leonard, J.C., Moscatello, D., Miller, L.C., Freking, B.A. 2008. Development of cell lines from the sheep used to construct the CHORI-243 ovine BAC library. Animal Biotechnology. 19(2):84-88.
- Bono, J.L., Keen, J.E., Clawson, M.L., Durso, L.M., Heaton, M.P., Laegreid, W.W. 2007. Association of Escherichia coli O157:H7 tir polymorphisms with human infection. BMC Infectious Diseases. 7:98.
- Miller, L.C., Harhay, G.P., Lager, K.M., Smith, T.P., Neill, J.D. 2008. Effect of porcine reproductive and respiratory syndrome virus on porcine alveolar macrophage function as determined using serial analysis of gene expression (SAGE). Developments in Biologicals. 132:169-174.
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Progress 10/01/06 to 09/30/07
Outputs
Progress Report Objectives (from AD-416) 1. Characterize the interaction of virus replication and macrophage responses. 2. Identify natural genetic variation associated with disease susceptibility. Approach (from AD-416) Identification of specific pathways that associate with variation in porcine reproductive and respiratory syndrome virus (PRRSV) replication and macrophage function leading to novel gene targets for the control of PRRSV infection. Alveolar macrophages will be obtained from diverse populations of swine and evaluated for their ability to support replication of PRRS viruses. Replication parameters will be estimated and macrophages that support either high or low levels of virus replication will be selected for studies of gene expression. Identifying PRRSV genotypes that confer fitness in macrophages, and host genes that respond to PRRSV fitness, to provide novel targets for intervention and control of PRRSV infections. These studies will use adapted isolates to identify viral genotypes that correlate with fitness of PRRSV in porcine alveolar macrophages and corresponding changes in macrophage transcriptional profiles. Genetic variation in specific ovine genes influences predisposition to ovine lentivirus (OLV) and the associated disease, ovine progressive pneumonia (OPP). We will thoroughly evaluate the most obvious candidate genomic regions for effects on lentiviral disease, like that containing CCR5. Our aim is to evaluate important regions of the genome for allelic association with the OLV disease susceptibility and progression phenotypes. Selection of regions will be based on a variety of scientific observations including, but not limited to, comparative mammalian biology. A selected set of 90 single nucleotide polymorphism (SNP) markers will be identified that are highly-informative in beef and dairy cattle. The development of this marker set represents non-hypothesis-driven research. The markers and genotyping assays for the markers will be readily available for any traceback needs. The same markers are also ideal for animal identification (i.e., sample matching) and routine parentage analysis. After ear tags and other physical identification devices have been removed, an animal�s DNA remains as a stable, accessible, integral, and identifiable component of its products and, thus, provides a gold standard for auditing the fidelity of physical labels and associated records. Accomplishments SNP markers for DNA-based traceback in North American beef and dairy cattle Scientists at USMARC developed a carefully selected set of 121 SNP markers that is useful for both "fingerprinting" animals and determining paternity. This includes 61 new highly-informative single nucleotide polymorphisms (SNPs) in U.S. beef and dairy populations that are publicly available in GenBank. These 121 SNP parentage markers are presently being used by companies in the U.S., Canada, and the EU. With these 121 parentage markers, the average probability that the genotypes from two unrelated individuals are identical by state is < 1E-50. The estimated average probability of paternity exclusion is < 0.9999999999 and 0.999999 for all 121 SNPs with one or zero parents known, respectively. This lends significant power to parentage-based traceback if needed. In many cases, parentage testing is the only form of traceback available in a disease outbreak. This was the case with the first U.S. BSE case reported in 2003. Many of these markers were used to trace the cow back to Canada. In total, more than 1000 SNPs have been identified, made publicly available, and transferred to more than 43 companies/institutions in more that 22 countries. Moreover, we have assisted more than a dozen commercial genotyping companies, forensic laboratories, research institutions, and universities in the adaptation of our assays to their particular genotyping platforms. The genetic tests include those for health, animal identity, parentage, gender, species, and prion diseases. National Program 103 - Animal Health National Program Component 3 - Countermeasures to prevent and control zoonotic diseases National Program Component 4 - Countermeasures to prevent and control respiratory diseases National Program Component 6 - Countermeasures to prevent and control enteric diseases National Program Component 8 - Countermeasures to prevent and control transmissible spongiform encephalopathies (TSE) diseases First set of PRRSV SAGE libraries constructed Porcine reproductive and respiratory syndrome virus (PRRSV) infection has been estimated to cost the U.S. pork industry over $500 million annually. Determining novel porcine and PRRSV gene or gene product targets for intervention and control of PRRSV infection will likely have a large impact on the U.S. and global pork industries. To determine these targets, changes in porcine and PRRSV gene product abundance upon PRRSV infection of porcine alveolar macrophages were characterized. Serial Analysis of Gene Expression (SAGE) was used to detect these changes. SAGE produced snapshots of the messenger RNA (gene product) population in PRRSV infected alveolar macrophages. From these snapshots, both the identity and abundance of messenger RNA was inferred from the 14 base SAGE tags captured from the end of the messenger RNA. Changes in tag abundance were observed in the PRRSV infected alveolar macrophages. Messenger RNA from more than 160 different porcine and 4 different PRRSV genes with significantly altered abundance levels were identified. The validity of SAGE inferred messenger RNA identity and abundance was evaluated and confirmed using alternative independent measures. National Program 103 - Animal Health National Program Component 2 - Genetic and biological determinants of disease susceptibility National Program Component 4 - Countermeasures to prevent and control respiratory diseases National Program Component 5 - Countermeasures to prevent and control reproductive and neonatal diseases PRRSV type I IFN and fitness screens Type I interferon expression in porcine alveolar macrophages infected with porcine reproductive and respiratory syndrome virus (PRRSV) causes highly significant losses to the swine industry worldwide. Productive infection occurs almost exclusively in cells of the monocyte-macrophage lineage both in vitro and in vivo, predominantly in alveolar macrophages of the lung. Thus, innate immune responses of the alveolar macrophages comprise the initial defense against PRRSV. Previously, we have demonstrated that PRRSV infection does not result in induction of type I interferons (IFN-alpha and -beta) by MARC-145 cells as expected with most RNA viruses. The results are significant because both IFN-alpha and -beta are products of the innate immune system--typically viewed as a result of the first response. Activation of this response signals other branches of the immune system to become activated and mount a protective response. The fact that PRRSV is capable of suppressing the activation of IFNs may explain the general delayed immune response to PRRSV infection. Here we report that differences in IFN induction elicited by PRRSV are responsible for differences in low growth and spread. Elucidation of the mechanism of PRRSV suppression of the type I IFN response may provide targets for novel vaccination approaches to control this important disease. National Program 103 - Animal Health National Program Component 2 - Genetic and biological determinants of disease susceptibility National Program Component 4 - Countermeasures to prevent and control respiratory diseases National Program Component 5 - Countermeasures to prevent and control reproductive and neonatal diseases Ovine progressive pneumonia (OPP) cases identified for genomic studies OPP is a slow, progressive and invariably fatal lymphoproliferative disease in sheep. It is associated with infection with ovine lentivirus (OLV), a common and endemic infection of U.S. sheep and has significant negative effects on sheep health and production. In Europe and other parts of the world, it is commonly referred to as maedi-visna virus (MVV) and may display lung or central nervous system (CNS) tropism. In North American sheep, it causes a diffuse interstitial pneumonia. Sheep may be infected at an early age but clinical disease is rarely observed before 3 years of age. Affected sheep have progressive weight loss and reduced production resulting in depressed pre-weaning growth of lambs and increasing culling risk. A number of reports have indicated that the sheep's genotype may predispose it to OLV infection and disease. Accordingly, USMARC scientists have defined three phenotypes and collected samples from more than 3000 sheep for genetic studies. The phenotypes included infection and disease resistance and rapid disease progression. These samples are now ready for genetic analyses. National Program 103 - Animal Health National Program Component 2 - Genetic and biological determinants of disease susceptibility National Program Component 4 - Countermeasures to prevent and control respiratory diseases Variation in ovine CCR5, an ortholog to a human lentivirus receptor The human lentivirus HIV-1 requires the CCR5 receptor for infection. People that lack CCR5 are resistant to HIV-1 infection and disease. To evaluate CCR5 as a potential candidate gene for influencing ovine lentivirus (OLV) infection and ovine progressive pneumonia (OPP), USMARC scientist identified variation in the ovine CCR5 coding sequence in diverse groups of U.S. sheep. These gene variants can now be evaluated with the above cases of OPP for influence on infection and disease. National Program 103 - Animal Health National Program Component 2 - Genetic and biological determinants of disease susceptibility National Program Component 4 - Countermeasures to prevent and control respiratory diseases Developed important animal cell lines for genomics research Cell lines from the sheep were used to construct the CHORI-243 ovine BAC library. Two cell lines, designated MARC.OVSM and MARC.OKF, were initiated from the aorta and kidney, respectively, obtained from the Texel ram used to make the CHORI-243 ovine BAC library. These cell lines have been submitted to the NIA Aging Cell Repository at the Coriell Cell Respositories, Camden, NJ, USA, and will be made publicly available. Fibroblast cells lines from the cow were used to sequence the bovine genome. Two cell lines, designated MARC.BGCF.2 and MARC.BGCF.1-3, were initiated from skin biopsies obtained from the Hereford cow whose DNA was used in sequencing the bovine genome. These cell lines were submitted to American Type Culture Collection (ATCC, Manassas, VA) and will be made publicly available. Because the amount of DNA required for genomic studies cannot be predicted, these cells will provide a relatively unlimited source of DNA for subsequent work. National Program 103 - Animal Health National Program Component 2 - Genetic and biological determinants of disease susceptibility Macrophage marker expression The BD FACScan flow cytometry system was setup to evaluate markers present on PRRSV-infected and control porcine macrophages. Protocols have also been developed to study macrophage marker expression immunohistochemically on fixed cytospin cell preparations of infected and control cells. National Program 103 - Animal Health National Program Component 2 - Genetic and biological determinants of disease susceptibility National Program Component 4 - Countermeasures to prevent and control respiratory diseases National Program Component 5 - Countermeasures to prevent and control reproductive and neonatal diseases
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