Genome Sequences of Mycobacteriophages Amgine, Amohnition, Bella96, Cain, DarthP, Hammy, Krueger, LastHope, Peanam, PhelpsODU, Phrank, SirPhilip, Slimphazie, and Unicorn

We report the genome sequences of 14 cluster K mycobacteriophages isolated using Mycobacterium smegmatis mc2155 as host. Four are closely related to subcluster K1 phages, and 10 are members of subcluster K6. The phage genomes span considerable sequence diversity, including multiple types of integrases and integration sites

Host Lipids Associated with Genome Replication in Flock House Virus

Positive-strand RNA [(+)RNA] viruses are significant human pathogens. A universal feature of (+)RNA viruses is that they replicate their genomes in association with host intracellular membranes. This association may be a target for broad spectrum antivirals against (+)RNA viruses. The (+)RNA virus we used for our studies is Flock House virus (FHV). FHV is a simple (+)RNA virus with a 4.5 kb bipartite genome that replicates in insect cells; Drosophilia melanogaster cells were used in this study. Previous work by others has shown an involvement of specific lipids, including phosphatidylcholine (PC), in FHV genome replication. To extend this work, we performed genetic knockouts of PC biosynthesis genes using Crispr/Cas9 genome editing. Lipid analysis indicated a reduction in total cellular PC content in these cells. Importantly, qPCR data showed a reduction in FHV RNA replication in cells where PC biosynthesis genes were knocked out. Next, we sought to more specifically examine the role of lipids in FHV genome replication by localizing lipids relative to sites of virus replication. We observed no significant enrichment of phosphatidylethanolamine (PE) or phosphatidylserine (PS) near or at sites of FHV replication. We are currently optimizing studies for imaging PC as well as lipid droplets. These initial studies have provided promising data regarding the role of specific lipids in (+)RNA virus replication

Specific Lipid Requirements and Localized Lipid Composition Changes Associated with Flock House Virus RNA Replication in Drosophila Cells

Positive-strand RNA [(+)RNA] viruses are significant human pathogens. A universal feature of (+)RNA viruses is that they replicate their genomes in association with host intracellular membranes. This association may be a target for broad spectrum antivirals against (+)RNA viruses. The (+)RNA virus used in our studies is the alphanodavirus Flock House virus (FHV). FHV is a simple (+)RNA virus with a 4.5 kb bipartite genome that replicates in insect cells. FHV replicates its RNA genome at the outer mitochondrial membrane of infected cells where it forms 50-70 nm in diameter invaginations that are membrane-bound RNA replication complexes. Previous work by others has shown an increase in the amount of phosphatidylcholine in FHV infected cells. Additionally, decreasing the amount of phosphatidylcholine in Drosophila cells thru targeted down regulation of biosynthetic genes decreased FHV replication. We hypothesized that increased levels of phosphatidylcholine were required for the formation and maintenance of the membrane-bound replication complexes located at mitochondria. To test this hypothesis, we synthesized the choline analog, propargylcholine, which is incorporated into phosphatidylcholine and can be “tagged” using copper(I) catalyzed cycloaddition chemistry. FHV infected cells were incubated with propargylcholine, tagged with a fluorescent dye, and visualized with confocal fluorescence microscopy. Our results suggest that there is an enrichment of phosphatidylcholine at the sites of FHV RNA genome replication. Additionally, we determined that two other major classes of cellular lipids, phosphatidylethanolamine and phosphatidylserine, were not enriched at FHV genome replication sites. Ongoing work includes a quantitative analysis of the mitochondrial lipid composition of FHV infected cells. Our work provides support for the importance of specific lipids in (+)RNA virus genome replication and the possibility that lipid biosynthetic pathways may be good antiviral targets

Isolation of 35 Mycobacteriophages and Genomic Analysis of the Novel Mycobacteriophage, Glass

Thirty-five new mycobacteriophages were isolated from soil samples collected on or nearby Hope College in Holland, Michigan. All were capable of infecting Mycobacterium smegmatis and produced a variety of plaque morphologies based on size, shape, and clarity, consistent with the isolation of an assortment of different phages. Both lytic and temperate phages appear represented in this collection. Purified phage stocks were used to prepare genomic DNA samples for restriction digest analysis. A comparison of those 35 digest results revealed few similarities among the group, further supporting our interpretation that most of the new phage isolates were distinct. One mycobacteriophage, Glass, was chosen for complete genome sequencing using the Illumina MiSeq platform and comparative genomic analysis. The predominant plaque produced by Glass at 32°C was turbid and 0.5-1.0mm in diameter, while plaque produced at 42°C was clear and 1.0-1.5mm in diameter. Genome sequence data for Glass revealed a relationship to a group of 12 mycobacteriophages in Cluster B2. The genome of Glass is 67.5 Kb, 69.0% GC, and contains 91 genes in agreement with the genome characteristics of closely related phage. A detailed analysis of the complete genome sequence and comparison with sequenced members of this small and unique group of mycobacteriophages is the subject of the second semester of this yearlong course and is presented

Ethical Challenges in the Treatment of Traumatized Refugees

The psychological plight of the large numbers of children and families who have immigrated to new lands has received increased professional attention. Among the multiple challenges confronting therapists who work with refugees, there is a growing need to be sensitive to ethical concerns unique to this population. This article addresses three representative ethical issues encountered in clinical work with traumatized refugees: the problem of informed consent, the resolution of conflicting cultural values, and the survivor's search for the meaning of inhumanity

Isolation of 18 Novel Mycobacteriophages and Genomic Analyses of Krueger and Phrappuccino

Eighteen new mycobacteriophages were isolated from soil samples collected around the state of Michigan and parts of the United States. All phages were capable of infecting Mycobacterium smegmatis and were isolated through either enrichment or direct plating at 25°C. A variety of plaque morphologies were produced based on size, shape, and clarity; both lytic and temperate phages appear represented in this collection. Two mycobacteriophages, Krueger and Phrappuccino, were chosen for complete genome sequencing and comparative genomic analyses. The predominant plaque produced by Krueger at 32°C was circular and 2 mm in diameter. The predominant plaque produced by Phrappuccino at 32°C was 1 mm in diameter, and took 48 hours to appear. Complete genome sequence for Krueger revealed relationships to members of the novel Subcluster K6, while Phrappuccino was not closely related to any known phage and is currently classified as a Singleton. The genome of Krueger is 60.3 Kb, 66.5% GC, and contains 101 genes, including 1 tRNA(Lys-TTT) gene; the genome of Phrappuccino is 136.3 Kb, 67.4% GC, and contains 200 genes. While Phrappuccino is a Singleton, there is strong evidence at the morphological (Myoviridae) and genomic levels for a relationship to Cluster C phages. Despite this relationship, Phrappuccino does not carry any tRNA genes. Forty (39.6%) and thirty-six (18%) protein coding genes were assigned functions in Krueger and Phrappuccino, respectively, based on comparative analyses. A detailed analysis of the complete genome sequences and comparison with sequenced mycobacteriophages is the subject of the second semester of this yearlong course and is presented