A Versatile Processing Workflow to Enable Pathogen Detection in Clinical Samples from Organs Using VIDISCA

Abstract

In recent years, refined molecular methods coupled with powerful high throughput sequencing technologies have increased the potential of virus discovery in clinical samples. However, host genetic material remains a complicating factor that interferes with discovery of novel viruses in solid tissue samples as the relative abundance of the virus material is low. Physical enrichment processing methods, although usually complicated, labor-intensive, and costly, have proven to be successful for improving sensitivity of virus detection in complex samples. In order to further increase detectability, we studied the application of fast and simple high-throughput virus enrichment methods on tissue homogenates. Probe sonication in high EDTA concentrations, organic extraction with Vertrel™ XF, or a combination of both, were applied prior to chromatography-like enrichment using Capto™ Core 700 resin, after which effects on virus detection sensitivity by the VIDISCA method were determined. Sonication in the presence of high concentrations of EDTA showed the best performance with an increased proportion of viral reads, up to 9.4 times, yet minimal effect on the host background signal. When this sonication procedure in high EDTA concentrations was followed by organic extraction with Vertrel™ XF and two rounds of core bead chromatography enrichment, an increase up to 10.5 times in the proportion of viral reads in the processed samples was achieved, with reduction of host background sequencing. We present a simple and semi-high-throughput method that can be used to enrich homogenized tissue samples for viral reads.

Dynamics of the Enteric Virome in a Swine Herd Affected by Non-PCV2/PRRSV Postweaning Wasting Syndrome

“…Viral metagenomics using unbiased next generation sequencing is a suitable technology to identify such virome changes, allowing us to increase our knowledge of virosphere diversity and providing us with a tool to elucidate the etiology of diseases the cause of which is yet unknown [7,8]. One of these unbiased virus discovery methods is VIDISCA, virus discovery cDNA-AFLP (amplified fragment length polymorphism) [9], which has been successfully used to detect a large number of novel human and animal viruses in cell culture supernatants [10], sera [11,12], feces [11,12], respiratory clinical samples [9], cerebrospinal fluid [13,14], and multiple solid tissues [15].…”

Special Issue “Molecular Detection and Typing of Viruses”

“…This virus was first identified by next generation sequencing [1]. One article in this special issue addresses the challenges of discovering new viruses by using next generation sequencing [2]. Following the spread of SARS-CoV-2 that rapidly caused a pandemic, it was then searched for and detected massively by reverse-transcription polymerase chain reaction (RT PCR).…”