Analysis Comparison for Rapid Identification of Pathogenic Virus from Infected Tissue Samples.

Junji Hosokawa-Muto,Yukiko Sassa-O’Brien,Yoshihito Fujinami,Hiroaki Nakahara

doi:10.3390/diagnostics12010196

Abstract

When examining infectious samples, rapid identification of the pathogenic agent is required for diagnosis and treatment or for investigating the cause of death. In our previous study, we applied exhaustive amplification using non-specific primers (the rapid determination system of viral genome sequences, the RDV method) to identify the causative virus via swab samples from a cat with a suspected viral infection. The purpose of the current study is to investigate suitable methods for the rapid identification of causative pathogens from infected tissue samples. First, the influenza virus was inoculated into mice to prepare infected tissue samples. RNA extracted from the mouse lung homogenates was transcribed into cDNA and then analyzed using the RDV method and next-generation sequencing, using MiSeq and MinION sequencers. The RDV method was unable to detect the influenza virus in the infected tissue samples. However, influenza virus reads were detected using next-generation sequencing. Comparing MiSeq and MinION, the time required for library and sequence preparation was shorter for MinION sequencing than for MiSeq sequencing. We conclude that when a causative virus needs to be rapidly identified from an infectious sample, MinION sequencing is currently the method of choice.

Highlights

When identifying a pathogenic virus from an infectious disease sample, the virus species is first estimated from the clinical course and the presenting symptoms of the patient, various tests and analyses of the pathological findings from the sample, and the application of methods such as immunochromatography, ELISA using a speciesspecific antibody, and PCR using species-specific primers [1]
Using cat fluid swab samples, we previously reported that the RDV method is useful for rapid virus identification in forensic samples from which it is difficult to estimate the virus species [3]
When a pathogenic virus needs to be rapidly identified from an infectious forensic sample, the RDV method allows for detection of the virus sequence with equipment widely available in most laboratories [3]

Summary

Introduction

When identifying a pathogenic virus from an infectious disease sample, the virus species is first estimated from the clinical course and the presenting symptoms of the patient, various tests and analyses of the pathological findings from the sample, and the application of methods such as immunochromatography, ELISA using a speciesspecific antibody, and PCR using species-specific primers [1]. In the case of bacterial agents, universal genes, such as the 16S ribosomal RNA gene and other genes common to bacteria, can be used for species estimation, but there are no such universal genes among viruses. If a virus species cannot be estimated, the extensive time and effort required to identify the virus can delay diagnosis and treatment or the determination of the cause of death. In such cases, exhaustive amplification using non-specific primers (the rapid determination system of viral genome sequences, RDV method) makes rapid virus identification possible [2]. Next-generation sequencing (NGS) has been successfully used for virus identification in a clinical context [4,5] and for the detection of novel viruses [6,7]

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Conclusion