SNPs for parentage testing and traceability in globally diverse breeds of sheep.

DNA-based parentage determination accelerates genetic improvement in sheep by increasing pedigree accuracy. Single nucleotide polymorphism (SNP) markers can be used for determining parentage and to provide unique molecular identifiers for tracing sheep products to their source. However, the utility of a particular "parentage SNP" varies by breed depending on its minor allele frequency (MAF) and its sequence context. Our aims were to identify parentage SNPs with exceptional qualities for use in globally diverse breeds and to develop a subset for use in North American sheep. Starting with genotypes from 2,915 sheep and 74 breed groups provided by the International Sheep Genomics Consortium (ISGC), we analyzed 47,693 autosomal SNPs by multiple criteria and selected 163 with desirable properties for parentage testing. On average, each of the 163 SNPs was highly informative (MAF≥0.3) in 48±5 breed groups. Nearby polymorphisms that could otherwise confound genetic testing were identified by whole genome and Sanger sequencing of 166 sheep from 54 breed groups. A genetic test with 109 of the 163 parentage SNPs was developed for matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. The scoring rates and accuracies for these 109 SNPs were greater than 99% in a panel of North American sheep. In a blinded set of 96 families (sire, dam, and non-identical twin lambs), each parent of every lamb was identified without using the other parent's genotype. In 74 ISGC breed groups, the median estimates for probability of a coincidental match between two animals (PI), and the fraction of potential adults excluded from parentage (PE) were 1.1×10(-39) and 0.999987, respectively, for the 109 SNPs combined. The availability of a well-characterized set of 163 parentage SNPs facilitates the development of high-throughput genetic technologies for implementing accurate and economical parentage testing and traceability in many of the world's sheep breeds.

Genetic testing for TMEM154 mutations associated with lentivirus susceptibility in sheep.

In sheep, small ruminant lentiviruses cause an incurable, progressive, lymphoproliferative disease that affects millions of animals worldwide. Known as ovine progressive pneumonia virus (OPPV) in the U.S., and Visna/Maedi virus (VMV) elsewhere, these viruses reduce an animal's health, productivity, and lifespan. Genetic variation in the ovine transmembrane protein 154 gene (TMEM154) has been previously associated with OPPV infection in U.S. sheep. Sheep with the ancestral TMEM154 haplotype encoding glutamate (E) at position 35, and either form of an N70I variant, were highly-susceptible compared to sheep homozygous for the K35 missense mutation. Our current overall aim was to characterize TMEM154 in sheep from around the world to develop an efficient genetic test for reduced susceptibility. The average frequency of TMEM154 E35 among 74 breeds was 0.51 and indicated that highly-susceptible alleles were present in most breeds around the world. Analysis of whole genome sequences from an international panel of 75 sheep revealed more than 1,300 previously unreported polymorphisms in a 62 kb region containing TMEM154 and confirmed that the most susceptible haplotypes were distributed worldwide. Novel missense mutations were discovered in the signal peptide (A13V) and the extracellular domains (E31Q, I74F, and I102T) of TMEM154. A matrix-assisted laser desorption/ionization-time-of flight mass spectrometry (MALDI-TOF MS) assay was developed to detect these and six previously reported missense and two deletion mutations in TMEM154. In blinded trials, the call rate for the eight most common coding polymorphisms was 99.4% for 499 sheep tested and 96.0% of the animals were assigned paired TMEM154 haplotypes (i.e., diplotypes). The widespread distribution of highly-susceptible TMEM154 alleles suggests that genetic testing and selection may improve the health and productivity of infected flocks.

The autoregulatory and transactivating functions of the human cytomegalovirus IE86 protein use independent mechanisms for promoter binding.

The functions of the human cytomegalovirus (HCMV) IE86 protein are paradoxical, as it can both activate and repress viral gene expression through interaction with the promoter region. Although the mechanism for these functions is not clearly defined, it appears that a combination of direct DNA binding and protein-protein interactions is involved. Multiple sequence alignment of several HCMV IE86 homologs reveals that the amino acids (534)LPIYE(538) are conserved between all primate and nonprimate CMVs. In the context of a bacterial artificial chromosome (BAC), mutation of both P535 and Y537 to alanines (P535A/Y537A) results in a nonviable BAC. The defective HCMV BAC does not undergo DNA replication, although the P535A/Y537A mutant IE86 protein appears to be stably expressed. The P535A/Y537A mutant IE86 protein is able to negatively autoregulate transcription from the major immediate-early (MIE) promoter and was recruited to the MIE promoter in a chromatin immunoprecipitation (ChIP) assay. However, the P535A/Y537A mutant IE86 protein was unable to transactivate early viral genes and was not recruited to the early viral UL4 and UL112 promoters in a ChIP assay. From these data, we conclude that the transactivation and repressive functions of the HCMV IE86 protein can be separated and must occur through independent mechanisms.

Inhibition of cellular DNA synthesis by the human cytomegalovirus IE86 protein is necessary for efficient virus replication.

Human cytomegalovirus (HCMV) expresses several proteins that manipulate normal cellular functions, including cellular transcription, apoptosis, immune response, and cell cycle control. The IE2 gene, which is expressed from the HCMV major immediate-early (MIE) promoter, encodes the IE86 protein. IE86 is a multifunctional protein that is essential for viral replication. The functions of IE86 include transactivation of cellular and viral early genes, negative autoregulation of the MIE promoter, induction of cell cycle progression from G0/G1 to G1/S, and arresting cell cycle progression at the G1/S transition in p53-positive human foreskin fibroblast (HFF) cells. Mutations were introduced into the IE2 gene in the context of the viral genome using bacterial artificial chromosomes (BACs). From these HCMV BACs, a recombinant virus (RV) with a single amino acid substitution in the IE86 protein was isolated that replicates slower and to lower titers than wild-type HCMV. HFF cells infected with the Q548R RV undergo cellular DNA synthesis and do not arrest at any point in the cell cycle. The Q548R RV is able to negatively autoregulate the MIE promoter, transactivate viral early genes, activate cellular E2F-responsive genes, and produce infectious virus. This is the first report of a viable recombinant HCMV that is unable to inhibit cellular DNA synthesis in infected HFF cells.

Improving baculovirus resistance to UV inactivation: increased virulence resulting from expression of a DNA repair enzyme.

The use of baculoviruses as biological control agents is hampered by their susceptibility to inactivation by ultraviolet (UV) light. In an attempt to reduce UV inactivation, an algal virus pyrimidine dimer-specific glycosylase, cv-PDG, was expressed in the baculovirus Autographa californica M nucleopolyhedrovirus (AcMNPV), and the infectivity of recombinant viruses expressing cv-PDG was measured after exposure to UV light. Expression of cv-PDG resulted in a 3-fold decrease in inactivation of budded virus by UV as measured by plaque assay in Spodoptera frugiperda Sf21 cells. However, occluded viruses expressing cv-PDG were not more resistant to UV inactivation than wild type AcMNPV when fed to either S. frugiperda or Trichoplusia ni neonate larvae. Surprisingly, however, viruses expressing cv-PDG showed a significant decrease in both the dose of occluded virus required for oral lethality and the time required for lethality compared to control virus, but these effects were only seen in S. frugiperda and not in T. ni larvae.