Multiplex PCR-Based Nanopore Sequencing and Epidemiological Surveillance of Hantaan orthohantavirus in Apodemus agrarius, Republic of Korea.

Whole-genome sequencing of infectious agents enables the identification and characterization of emerging viruses. The MinION device is a portable sequencer that allows real-time sequencing in fields or hospitals. Hantaan orthohantavirus (Hantaan virus, HTNV), harbored by Apodemus agrarius, causes hemorrhagic fever with renal syndrome (HFRS) and poses a critical public health threat worldwide. In this study, we aimed to evaluate the feasibility of using nanopore sequencing for whole-genome sequencing of HTNV from samples having different viral copy numbers. Amplicon-based next-generation sequencing was performed in A. agrarius lung tissues collected from the Republic of Korea. Genomic sequences of HTNV were analyzed based on the viral RNA copy numbers. Amplicon-based nanopore sequencing provided nearly full-length genomic sequences of HTNV and showed sufficient read depth for phylogenetic analysis after 8 h of sequencing. The average identity of the HTNV genome sequences for the nanopore sequencer compared to those of generated from Illumina MiSeq revealed 99.8% (L and M segments) and 99.7% (S segment) identities, respectively. This study highlights the potential of the portable nanopore sequencer for rapid generation of accurate genomic sequences of HTNV for quicker decision making in point-of-care testing of HFRS patients during a hantavirus outbreak.

A Clinical Case of Scrub Typhus in the United States Forces Korea Patient with Eschar and Genetic Identification of Orientia tsutsugamushi Using Multiplex PCR-Based Next-Generation Sequencing.

An epidemiological investigation was conducted for a scrub typhus case reported in a U.S. Forces Korea military patient in the Republic of Korea in November 2018. The patient had a fever, maculopapular rash, and an eschar. The full-length sequence of Orientia tsutsugamushi 56-kDa type-specific antigen (TSA) gene was obtained from eschar tissue by multiplex PCR-based Next Generation Sequencing for genetic identification. Based on the 56-kDa TSA gene, the O. tsutsugamushi aligned most closely with the Boryong strain.

Active Targeted Surveillance to Identify Sites of Emergence of Hantavirus.

BACKGROUND: Endemic outbreaks of hantaviruses pose a critical public health threat worldwide. Hantaan orthohantavirus (HTNV) causes hemorrhagic fever with renal syndrome (HFRS) in humans. Using comparative genomic analyses of partial and nearly complete sequences of HTNV from humans and rodents, we were able to localize, with limitations, the putative infection locations for HFRS patients. Partial sequences might not reflect precise phylogenetic positions over the whole-genome sequences; finer granularity of rodent sampling reflects more precisely the circulation of strains. METHODS: Five HFRS specimens were collected. Epidemiological surveys were conducted with the patients during hospitalization. We conducted active surveillance at suspected HFRS outbreak areas. We performed multiplex polymerase chain reaction-based next-generation sequencing to obtain the genomic sequence of HTNV from patients and rodents. The phylogeny of human- and rodent-derived HTNV was generated using the maximum likelihood method. For phylogeographic analyses, the tracing of HTNV genomes from HFRS patients was defined on the bases of epidemiological interviews, phylogenetic patterns of the viruses, and geographic locations of HTNV-positive rodents. RESULTS: The phylogeographic analyses demonstrated genetic clusters of HTNV strains from clinical specimens, with HTNV circulating in rodents at suspected sites of patient infections. CONCLUSIONS: This study demonstrates a major shift in molecular epidemiological surveillance of HTNV. Active targeted surveillance was performed at sites of suspected infections, allowing the high-resolution phylogeographic analysis to reveal the site of emergence of HTNV. We posit that this novel approach will make it possible to identify infectious sources, perform disease risk assessment, and implement preparedness against vector-borne viruses.

Detection of single-base mutation in RNA using T4 RNA ligase-based nick-joining or DNAzyme-based nick-generation.

A single nucleotide polymorphism (SNP) is a common genetic variation when a single nucleotide differs between members of a species or paired chromosome. Due to its association with disease susceptibility and drug resistance, SNP detection is of great value in studying the variation in drug responses. Here we present two quantitative SNP detection methods for a single-base mismatch in RNA, based on nick-joining and nick-generating activities of T4 RNA ligase and DNAzyme, respectively. T4 RNA ligase successfully discriminated a one-base mismatch in the ligation junction, and the designed DNAzyme cleaved RNA by discerning a single-base mismatch in the cleaving site.

Cooperative translocation enhances the unwinding of duplex DNA by SARS coronavirus helicase nsP13.

SARS coronavirus encodes non-structural protein 13 (nsP13), a nucleic acid helicase/NTPase belonging to superfamily 1 helicase, which efficiently unwinds both partial-duplex RNA and DNA. In this study, unwinding of DNA substrates that had different duplex lengths and 5'-overhangs was examined under single-turnover reaction conditions in the presence of excess enzyme. The amount of DNA unwound decreased significantly as the length of the duplex increased, indicating a poor in vitro processivity. However, the quantity of duplex DNA unwound increased as the length of the single-stranded 5'-tail increased for the 50-bp duplex. This enhanced processivity was also observed for duplex DNA that had a longer single-stranded gap in between. These results demonstrate that nsP13 requires the presence of a long 5'-overhang to unwind longer DNA duplexes. In addition, enhanced DNA unwinding was observed for gapped DNA substrates that had a 5'-overhang, indicating that the translocated nsP13 molecules pile up and the preceding helicase facilitate DNA unwinding. Together with the propensity of oligomer formation of nsP13 molecules, we propose that the cooperative translocation by the functionally interacting oligomers of the helicase molecules loaded onto the 5'-overhang account for the observed enhanced processivity of DNA unwinding.

Isothermal DNA amplification in vitro: the helicase-dependent amplification system.

Since the development of polymerase chain reaction, amplification of nucleic acids has emerged as an elemental tool for molecular biology, genomics, and biotechnology. Amplification methods often use temperature cycling to exponentially amplify nucleic acids; however, isothermal amplification methods have also been developed, which do not require heating the double-stranded nucleic acid to dissociate the synthesized products from templates. Among the several methods used for isothermal DNA amplification, the helicase-dependent amplification (HDA) is discussed in this review with an emphasis on the reconstituted DNA replication system. Since DNA helicase can unwind the double-stranded DNA without the need for heating, the HDA system provides a very useful tool to amplify DNA in vitro under isothermal conditions with a simplified reaction scheme. This review describes components and detailed aspects of current HDA systems using Escherichia coli UvrD helicase and T7 bacteriophage gp4 helicase with consideration of the processivity and efficiency of DNA amplification.