Microfluidic PCR Amplification and MiSeq Amplicon Sequencing Techniques for High-Throughput Detection and Genotyping of Human Pathogenic RNA Viruses in Human Feces, Sewage, and Oysters.

Mamoru Oshiki,Takashi Yamaguchi,Hideki Harada,Tsutomu Okubo,Masashi Hatamoto,Daisuke Sano,Shigeki Uemura,Satoshi Ishii,Kengo Kubota,Takahiro Segawa,Akinori Iguchi,Tadashi Tagawa,Shinobu Kazama,Naohiro Kobayashi,Takayuki Miura,Nobuo Araki

doi:10.3389/fmicb.2018.00830

Abstract

Detection and genotyping of pathogenic RNA viruses in human and environmental samples are useful for monitoring the circulation and prevalence of these pathogens, whereas a conventional PCR assay followed by Sanger sequencing is time-consuming and laborious. The present study aimed to develop a high-throughput detection-and-genotyping tool for 11 human RNA viruses [Aichi virus; astrovirus; enterovirus; norovirus genogroup I (GI), GII, and GIV; hepatitis A virus; hepatitis E virus; rotavirus; sapovirus; and human parechovirus] using a microfluidic device and next-generation sequencer. Microfluidic nested PCR was carried out on a 48.48 Access Array chip, and the amplicons were recovered and used for MiSeq sequencing (Illumina, Tokyo, Japan); genotyping was conducted by homology searching and phylogenetic analysis of the obtained sequence reads. The detection limit of the 11 tested viruses ranged from 100 to 103 copies/μL in cDNA sample, corresponding to 101–104 copies/mL-sewage, 105–108 copies/g-human feces, and 102–105 copies/g-digestive tissues of oyster. The developed assay was successfully applied for simultaneous detection and genotyping of RNA viruses to samples of human feces, sewage, and artificially contaminated oysters. Microfluidic nested PCR followed by MiSeq sequencing enables efficient tracking of the fate of multiple RNA viruses in various environments, which is essential for a better understanding of the circulation of human pathogenic RNA viruses in the human population.

Highlights

Various species of human RNA viruses, including Aichi virus (AiV), astrovirus (AstV), enterovirus (EV), hepatitis A virus (HAV), hepatitis E virus (HEV), norovirus (NoV), rotavirus (RV), sapovirus (SaV), and human parechovirus (HPeV), have been recognized as causative agents of waterborne viral gastroenteritis and hepatitis (Bosch, 1998; Harvala et al, 2008; Sinclair et al, 2009; Pham et al, 2010; Svraka et al, 2010; Drexler et al, 2011)
When the detection limit was defined as 10 sequence reads per sample, each tested virus was detectable in the following dilution series: 102 for AiV, 102 for AstV, 101 for EV, for NoV genogroup I (GI), for NoV GII, 102 for NoV GIV, 101 for HAV, 100 for HEV, 102 for RV, 103 for SaV, and 102 for HPeV
By considering dilution factors resulting from the processes of virus concentration, RNA extraction, and cDNA synthesis, the detection limits were calculated to be 101–104 copies/mL for sewage, 105–108 copies/g-human feces, and 102–105 copies/g-digestive tissues of oysters

Summary

Introduction

Various species of human RNA viruses, including Aichi virus (AiV), astrovirus (AstV), enterovirus (EV), hepatitis A virus (HAV), hepatitis E virus (HEV), norovirus (NoV), rotavirus (RV), sapovirus (SaV), and human parechovirus (HPeV), have been recognized as causative agents of waterborne viral gastroenteritis and hepatitis (Bosch, 1998; Harvala et al, 2008; Sinclair et al, 2009; Pham et al, 2010; Svraka et al, 2010; Drexler et al, 2011). NoV is composed of seven genogroups, and each genogroup is further subdivided into genotypes (Vinjé,, 2015). These RNA viruses circulate in aquatic environments and the human population (Iwai et al, 2009; Sano et al, 2011; Hata et al, 2015; Miura et al, 2016; Pu et al, 2016). The viruses exist at high concentration in feces of patients and are released into sewage system (Lee et al, 2007; Drexler et al, 2011). Sewage effluents still contain the viruses at certain level (Miura et al, 2015; Kobayashi et al, 2017), which contaminate natural water resources. For a better understanding of the circulation and prevalence of RNA viruses and for identification of a contamination source, detection and genotyping of multiple viruses are essential (Kazama et al, 2017)

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Conclusion