2011 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.
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.
Discovery of a gene for pneumonia susceptibility in sheep. Ovine progressive pneumonia is an incurable slow-acting disease of sheep caused by persistent lentivirus infection. The Animal and Plant Health Inspection 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 infection 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 are significantly less likely to become infected.
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: http://downloads.hindawi.com/journals/av/2010/864181.pdf.
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.