Tracking ebolavirus genomic drift with a resequencing microarray

Abstract

Filoviruses are emerging pathogens that cause acute fever with high fatality rate and present a global public health threat. During the 2013–2016 Ebola virus outbreak, genome sequencing allowed the study of virus evolution, mutations affecting pathogenicity and infectivity, and tracing the viral spread. In 2018, early sequence identification of the Ebolavirus as EBOV in the Democratic Republic of the Congo supported the use of an Ebola virus vaccine. However, field-deployable sequencing methods are needed to enable a rapid public health response. Resequencing microarrays (RMA) are a targeted method to obtain genomic sequence on clinical specimens rapidly, and sensitively, overcoming the need for extensive bioinformatic analysis. This study presents the design and initial evaluation of an ebolavirus resequencing microarray (Ebolavirus-RMA) system for sequencing the major genomic regions of four Ebolaviruses that cause disease in humans. The design of the Ebolavirus-RMA system is described and evaluated by sequencing repository samples of three Ebolaviruses and two EBOV variants. The ability of the system to identify genetic drift in a replicating virus was achieved by sequencing the ebolavirus glycoprotein gene in a recombinant virus cultured under pressure from a neutralizing antibody. Comparison of the Ebolavirus-RMA results to the Genbank database sequence file with the accession number given for the source RNA and Ebolavirus-RMA results compared to Next Generation Sequence results of the same RNA samples showed up to 99% agreement.

Multiplex detection and identification of viral, bacterial, and protozoan pathogens in human blood and plasma using an expanded high-density resequencing microarray platform

“…The code for the bbp_i2o pipeline and construction of TVRSD is freely available at: https://github.com/FDA/Resequencing-Microarray-Pipeline/blob/main/bbp_i2o_2 . As previously described ( Tiper et al, 2022 ), a very simple graphic user interface allows the choice of the pipeline and the choice of the FASTA file. With a click on the “run” button, the bbp_i2o executes a series of PERL-based scripts that link together some minor calculations and open-source software: BLAST for database searching ( Altschul et al, 1990 ) and MUSCLE for multiple sequence alignment ( Edgar, 2004 ), all unseen by the operator.…”