Broad Virus Detection and Variant Discovery in Fecal Samples of Hematopoietic Transplant Recipients Using Targeted Sequence Capture Metagenomics

Abstract

Pediatric allogeneic hematopoietic stem cell transplantation (HSCT) patients often suffer from gastro-intestinal (GI) disease caused by viruses, Graft-versus-Host Disease (GVHD) or a combination of the two. Currently, the GI eukaryotic virome of HSCT recipients remains relatively understudied, which complicates the understanding of its role in GVHD pathogenicity. As decisions regarding immunosuppressive therapy in the treatment of virus infection or GVHD, respectively, can be completely contradicting, it is crucial to better understand the prevalence and relevance of viruses in the GI tract in the HSCT setting. A real time PCR panel for a set of specific viruses widely used to diagnose the most common causes of GI viral gastroenteritis is possibly insufficient to grasp the full extent of viruses present. Therefore, we applied the targeted sequence capture method ViroCap to residual fecal samples of 11 pediatric allogeneic HSCT recipients with GI symptoms and a suspicion of GVHD, to enrich for nucleic acids of viruses that are known to infect vertebrate hosts. After enrichment, NGS was applied to broadly detect viral sequences. Using ViroCap, we were able to detect viruses such as norovirus and adenovirus (ADV), that had been previously detected using clinical diagnostic PCR on the same sample. In addition, multiple, some of which clinically relevant viruses were detected, including ADV, human rhinovirus (HRV) and BK polyomavirus (BKV). Interestingly, in samples in which specific PCR testing for regular viral GI pathogens did not result in a diagnosis, the ViroCap pipeline led to the detection of viral sequences of human herpesvirus (HHV)-7, BKV, HRV, KI polyomavirus and astrovirus. The latter was an only recently described variant and showed extensive sequence mismatches with the applied real time PCR primers and would therefore not have been detected if tested. Our results indicate that target enrichment of viral nucleic acids through ViroCap leads to sensitive and broad possibly clinically relevant virus detection, including the detection of newer variants in clinical HSCT recipient samples. As such, ViroCap could be a useful detection tool clinically, but also in studying the associations between viral presence and GVHD.

Hybrid-Capture Target Enrichment in Human Pathogens: Identification, Evolution, Biosurveillance, and Genomic Epidemiology

“…Therefore, an early panel, Virocap, used 337 viral species genomes as a reference for bait design [ 113 ]. The Virocap panel comprised approximately 2 million baits and was applied to a study detecting gastrointestinal (GI) viruses in transplant recipients for hematopoietic stem cells, specifically to differentiate between graft-versus-host disease or GI disease caused by viral pathogens [ 114 ]. Common culprits such as norovirus (see also [ 115 ]) and adenovirus positive in PCR diagnostics were also positive using HC, but a diversity of clinically relevant viruses, especially in those without any positive PCR result for common GI disease viral pathogens, were also captured, once again highlighting the need for judicious interpretation of results is required when using CM as a possible avenue for diagnosis.…”

Possible role of intestinal human viruses and bacteriophages following hematopoietic stem cell transplantation: a mini-review

“…Moreover, decreased detection rates were noted for bacteriophages, except for Siphoviridae. A similar work was carried out by a Dutch group [13]. The authors used the original ViroCap system to study fecal samples, with a large panel of probes for 34 families of DNA and RNA viruses (a total of 337 species).…”

Comparison of the performance of two targeted metagenomic virus capture probe-based methods using synthetic viral sequences and clinical samples

“…Moreover, these earlier data showed that this hybridisation panel of approximately two million capture probes designed in 2015 was suited for the detection of novel coronaviruses by reactivity with other vertebrate betacoronavirus probes 10 . During the past years, this specific hybridisation panel distributed by Roche has been adopted in a broad range of different clinical 15,[22][23][24][25][26][27][28][29] and zoonotic settings 22,29,30 is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review)…”