2007 Annual Report 1a.Objectives (from AD-416) 1. Characterize the interaction of virus replication and macrophage responses. 2. Identify natural genetic variation associated with disease susceptibility. 1b.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. 3.Progress Report None 4.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 5.Significant Activities that Support Special Target Populations None 6.Technology Transfer