Whole-genome Sequencing of SARS-CoV-2: Using Phylogeny and Structural Modeling to Contextualize Local Viral Evolution.

INTRODUCTION: The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has created a global pandemic resulting in over 1 million deaths worldwide. In the Department of Defense (DoD), over 129,000 personnel (civilians, dependents, and active duty) have been infected with the virus to date. Rapid estimations of transmission and mutational patterns of virus outbreaks can be accomplished using whole-genome viral sequencing. Deriving interpretable and actionable results from pathogen sequence data is accomplished by the construction of phylogenetic trees (from local and global virus sequences) and by the creation of protein maps, to visualize and predict the effects of structural protein amino acid mutations. MATERIALS AND METHODS: We developed a sequencing and bioinformatics workflow for molecular epidemiological SARS-CoV-2 surveillance using excess clinical specimens collected under an institutional review board exempt protocol at Joint Base San Antonio, Lackland AFB. This workflow includes viral RNA isolation, viral load quantification, tiling-based next-generation sequencing, sequencing and bioinformatics analysis, and data visualization via phylogenetic trees and protein mapping. RESULTS: Sequencing of 37 clinical specimens collected at JBSA/Lackland revealed that by June 2020, SAR-CoV-2 strains carrying the 614G mutation were the predominant cause of local coronavirus disease 2019 infections. We identified 109 nucleotide changes in the coding region of the SARS-CoV-2 genome (which lead to 63 unique, non-synonymous amino acid mutations), one mutation in the 5'-untranslated region (UTR), and two mutations in the 3'UTR. Furthermore, we identified and mapped six additional spike protein amino acid changes-information which could potentially aid vaccine design. CONCLUSION: The workflow presented here is designed to enable DoD public health officials to track viral evolution and conduct near real-time evaluation of future outbreaks. The generation of molecular epidemiological sequence data is critical for the development of disease intervention strategies-most notably, vaccine design. Overall, we present a streamlined sequencing and bioinformatics methodology aimed at improving long-term readiness efforts in the DoD.

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay.

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are the main branches of innate immunity, and many of the factors regulating these responses are evolutionarily conserved between invertebrates and mammals. Phagocytosis, the central component of cellular innate immunity, is carried out by specialized blood cells of the immune system. The fruit fly, Drosophila melanogaster, has emerged as a powerful genetic model to investigate the molecular mechanisms and physiological impacts of phagocytosis in whole animals. Here we demonstrate an injection-based in vivo phagocytosis assay to quantify the particle uptake and destruction by Drosophila blood cells, hemocytes. The procedure allows researchers to precisely control the particle concentration and dose, making it possible to obtain highly reproducible results in a short amount of time. The experiment is quantitative, easy to perform, and can be applied to screen for host factors that influence pathogen recognition, uptake, and clearance.

The Splicing Factor RNA-Binding Fox Protein 1 Mediates the Cellular Immune Response in Drosophila melanogaster.

The uptake and destruction of bacteria by phagocytic cells is an essential defense mechanism in metazoans. To identify novel genes involved in the phagocytosis of Staphylococcus aureus, a major human pathogen, we assessed the phagocytic capacity of adult blood cells (hemocytes) of the fruit fly, Drosophila melanogaster, by testing several lines of the Drosophila Genetic Reference Panel. Natural genetic variation in the gene RNA-binding Fox protein 1 (Rbfox1) correlated with low phagocytic capacity in hemocytes, pointing to Rbfox1 as a candidate regulator of phagocytosis. Loss of Rbfox1 resulted in increased expression of the Ig superfamily member Down syndrome adhesion molecule 4 (Dscam4). Silencing of Dscam4 in Rbfox1-depleted blood cells rescued the fly's cellular immune response to S. aureus, indicating that downregulation of Dscam4 by Rbfox1 is critical for S. aureus phagocytosis in Drosophila To our knowledge, this study is the first to demonstrate a link between Rbfox1, Dscam4, and host defense against S. aureus.

A glutamate-dependent redox system in blood cells is integral for phagocytosis in Drosophila melanogaster.

Glutamate transport is highly regulated as glutamate directly acts as a neurotransmitter and indirectly regulates the synthesis of antioxidants. Although glutamate deregulation has been repeatedly linked to serious human diseases such as HIV infection and Alzheimer's, glutamate's role in the immune system is still poorly understood. We find that a putative glutamate transporter in Drosophila melanogaster, polyphemus (polyph), plays an integral part in the fly's immune response. Flies with a disrupted polyph gene exhibit decreased phagocytosis of microbial-derived bioparticles. When infected with S. aureus, polyph flies show an increase in both susceptibility and bacterial growth. Additionally, the expression of two known glutamate transporters, genderblind and excitatory amino acid transporter 1, in blood cells affects the flies' ability to phagocytose and survive after an infection. Consistent with previous data showing a regulatory role for glutamate transport in the synthesis of the major antioxidant glutathione, polyph flies produce more reactive oxygen species (ROS) as compared to wild-type flies when exposed to S. aureus. In conclusion, we demonstrate that a polyph-dependent redox system in blood cells is necessary to maintain the cells' immune-related functions. Furthermore, our model provides insight into how deregulation of glutamate transport may play a role in disease.

Phospholipase C beta 4 in mouse hepatocytes: rhythmic expression and cellular distribution.

BACKGROUND: Circadian regulated physiological processes have been well documented in the mammalian liver. Phospholipases are important mediators of both cytoplasmic and nuclear signaling mechanisms in hepatocytes, and despite a potentially critical role for these enzymes in regulating the temporal aspect of hepatic physiology, their involvement in the circadian liver clock has not been the subject of much investigation. The phospholipase C beta4 (PLCbeta4) enzyme is of particular interest as it has been linked to circadian clock function. In general, there is no knowledge of the role of the PLCbeta4 isozyme in mammalian hepatocytes as this is the first report of its expression in the mammalian liver. RESULTS: We found that in the liver of mice housed on a light:dark cycle, PLCbeta4 protein underwent a significant circadian rhythm with a peak occurring during the early night. In constant darkness, the protein rhythm was more robust and peaked around dusk. We also observed a significant oscillation in plcbeta4 gene expression in the livers of mice housed in both photoperiodic and constant dark conditions. The cellular distribution of the protein in hepatocytes varied over the course of the circadian day with PLCbeta4 primarily cytoplasmic around dusk and nuclear at dawn. CONCLUSION: Our results indicate that PLCbeta4 gene and protein expression is regulated by a circadian clock in the mouse liver and is not dependent on the external photoperiod. A light-independent daily translocation of PLCbeta4 implies that it may play a key role in nuclear signaling in hepatocytes and serve as a daily temporal cue for physiological processes in the liver.