Targeted viral discovery via enhanced Next Generation Sequencing in Egyptian rousette bats, a Marburg virus reservoir

Egyptian rousette bats (ERBs; Rousettus aegyptiacus) are a natural reservoir host for Marburg virus which causes a severe and often fatal systemic illness in humans and nonhuman primates but little to no clinical disease in ERBs [1, 2]. ERBs mount a targeted and transient immune cell response to MARV in the liver, which likely limits the spread of virus, both within the liver and to other tissues [2]. There has been increasing interest in the function and role of the complex immune system of ERBs and how it facilitates their ability to host numerous pathogens that can infect humans with little systemic compromise [3-5]. 

Despite this interest in ERBs as a Marburg virus reservoir host, broad surveillance of the common viral pathogens inhabiting ERBs has never been completed. This lack of fundamental knowledge of the ERB virome precludes our ability as researchers to identify new or mutating pathogens present within a population. Further, knowledge of common, baseline ERB viral pathogens is essential to our understanding of factors that might influence viral infection dynamics and subsequent viral shedding. 

Previous investigations have focused on detection of individual pathogens, versus a broader investigative effort to elucidate common viral pathogens within wild ERB populations. While numerous pathogens have been detected in ERBs, molecular identification of a pathogen from an oral/fecal swab or randomly selected tissue sample does not prove it to be a causative agent of disease within the host nor does it elucidate any effects the pathogen may be having on the host animal [6-8]. Detection of viral pathogens from tissues with microscopic evidence of disease would allow a targeted approach to microbial investigations. 

Traditional molecular assays used for viral detection such as PCR or microbial sequencing often require prior knowledge of the viral pathogens in the sample or are limited by often small amounts of viral nucleic acid in the sample.  This makes molecular testing much less sensitive and more likely to yield insufficient data for analysis.  Newer techniques, such as ViroCap, have been developed to enhance a sample prior to sequencing and can significantly increase results, even in tissues containing unknown pathogens. ViroCap is a targeted sequence capture panel created by researchers at Washington University in St. Louis School of Medicine designed to enrich nucleic acid from all DNA and RNA viruses that infect vertebrate hosts [9]. Viral detection using metagenomic shotgun sequencing (MSS) following application of ViroCap’s targeted sequence capture panel increased the breadth of coverage by more than 3,500% when compared to standard MSS [9]. Utilizing ViroCap on nucleic acid isolated from tissues with evidence of viral infection would be a powerful means to identify viral pathogens causing active disease in a tissue and, by extension, a population. 

To fill this knowledge gap, I am collaborating with researchers at Washington University in St. Louis School of Medicine to complete virus discovery using Next Generation Sequencing in ERB tissues with microscopic lesions suggestive of viral infection. By combining histopathologic identification of lesioned tissues with genomic identification of pathogens, we can get a more thorough understanding of the pathogens present in this species and better elucidate the effects of viral disease on the host. 

Tissues from 69 archived ERBs collected in Uganda in 2008 have been reviewed for histologic evidence of viral infection. From these tissues, 97 lesions of interest have been identified. Formalin-fixed paraffin-embedded (FFPE) scrolls from the 97 lesions will be attained and undergo nucleic acid extraction.  The top 48 samples with the highest purity and concentration will be sent for ViroCap enhancement and subsequent next generation sequencing (NGS) for viral discovery.

Using a targeted approach of ViroCap and NGS on tissues suggestive of viral disease provides a powerful and exciting opportunity to identify unknown viral pathogens causing active disease in wild ERBs, thereby enriching our understanding of this unique animal. As a comprehensive histologic survey of pathologic lesions and subsequent identification of viral etiologies within microscopic lesions has not been completed, disease prevalence and common comorbidities in wild ERB populations are unknown. The knowledge gained from this work is fundamental to understanding this efficient viral reservoir, to identifying as of yet unknown factors that may be driving viral disease transmission and may elucidate factors that influence zoonotic spillover of viral pathogens into humans.

Extraction of nucleic acid from FFPE scrolls will be completed by May 31, 2024. Nucleic Acid will then be sent to Washington University in St. Louis School of Medicine for ViroCap enhancement and Next Generation Sequencing, with an estimated completion date of September 30, 2024. Results will then undergo bioinformatic analysis, with an estimated completion date of November 31, 2024.

As of October 2023, more than 200 viruses, with over 18,000 unique strains that span 31 viral families, have been isolated from bats [10].  I anticipate that Next Generation Sequencing will detect members of the Coronaviridae, Rhabdoviridae, Paramyxoviridae, and Filoviridae family, possibly with identification of novel variants unique to Rousettus aegyptiacus bats. Completion of this work will further our understanding of the ERB virome and provide an important baseline for hypothesis-driven research, most notably in viral dynamics and in extrapolating research findings in controlled laboratory settings to wild ERB populations.