www.cdc.gov | Contact CDC at: 1-800-CDC-INFO or www.cdc.gov/info The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention. Contact Info Using Nextstrain to Visualize Genomic Data from the 2022 Sudan Ebolavirus Outbreak in Uganda Stephen Balinandi1 , Shannon Whitmer2 , Sophia Mulei1 , Angella C. Nassuna1 , Markus Kainulainen2 , Elizabeth Shedroff2 , Inna Krapiunaya2 , Florine Scholte2 , Luke Nyakarahuka1 , Alex Tumusiime1 , Jackson Kyondo1 , Jimmy Baluku1 , Jocelyn Kiconco1 , Julius Lutwama1 , Trevor R. Shoemaker2 , Joel Montgomery2 , Pontiano Kaleebu1 , John D. Klena2 1. Uganda Virus Research Institute, Entebbe, Uganda 2. Viral Special Pathogens Branch, Centers for Disease Control and Prevention, Atlanta, Georgia, USA Elizabeth Shedroff ubu2@cdc.gov +1 330-888-3924 Nextstrain is a versatile tool to analyze, visualize, and interpret pathogen genome data and remains relevant in a multitude of applications to study SUDV. Introduction Ebolaviruses are single-stranded negative-sense RNA (ssRNA) viruses in the Filovirus family; Sudan ebolavirus (Orthoebolavirus sudanense) represents one species of this family. They are composed of seven viral proteins: nucleoprotein (NP), polymerase complex component (VP35), matrix protein (VP40), glycoprotein (GP), transcription activator (VP30), second matrix protein (VP42), and polymerase (L). The Ebolavirus genome contains a transcriptional stutter allowing the GP gene to encode a dimeric structural glycoprotein, secreted glycoprotein (sGP) and small nonstructural sGP (ssGP), and full-length GP. Ebolavirus is a type of viral hemorrhagic fever (VHF) with an incubation period of 2-21 days. Nextstrain is a publicly available bioinformatics tool for phylogenetic analysis and data visualization of genomic pathogen data. It is powered by a collection of modules called Augur that analyze the genome data and turns it into a readable dataset for Auspice, a server for visualization. Nextstrain’s dashboard allows a user to view phylogenetic trees, phylogeographic maps, and mutation prevalence. During the outbreak, 142 confirmed and 22 probable cases were officially identified. Next-generation sequencing (NGS) was performed at the Uganda Virus Research Institute (UVRI) Viral Hemorrhagic Fever Lab using RNA extracted from confirmed cases by either an unbiased method on Illumina technologies, or an amplicon-based method using Oxford Nanopore technologies. Overall, UVRI attempted sequencing on 129 specimens and generated 120 genomes with greater than 90% coverage from 114 unique cases which represent approximately 80% of confirmed cases. As outbreak data were collected, naturally occurring genotypic differences between Mubende cases and other districts became apparent, indicating mutation over time. Uses of these data include: 1. Delineating unknown epidemiological linkages or transmission events 2. Using the calculated intra-outbreak mutation rate to help characterize the source of a future re-emergence event. 3. Visualizing mutation prevalence and the potential impact on vaccine efficacy (Kabakli, 2019) Figure 1. Phylogeographic map (left) and inferred phylogenetic relationships (right) of 120 of the 2022 SUDV sequences. Phylogenetic relationships were determined by Random Axelerated Maximum Likelihood. District location is represented by color. Figure 2.Inferred intra-outbreak mutation rate. District location is represented by color. Rate estimate is expressed by a black line. Figure 3. Phylogenetic relationships between 2022 SUDV sequences (top) and entropy of amino acid mutations from the SUDV sequences (bottom). An R -> C mutation at NP711 is highlighted in green. It is shown in the tree via blue coloring for the Arg genotype and yellow coloring for the Cys genotype.