Phylogeographic diversity and hybrid zone of Hantaan orthohantavirus collected in Gangwon Province, Republic of Korea

Abstract

Background Hantaan orthohantavirus (Hantaan virus, HTNV), harbored by Apodemus agrarius (the striped field mouse), causes hemorrhagic fever with renal syndrome (HFRS) in humans. Viral genome-based surveillance at new expansion sites to identify HFRS risks plays a critical role in tracking the infection source of orthohantavirus outbreak. In the Republic of Korea (ROK), most studies demonstrated the serological prevalence and genetic diversity of orthohantaviruses collected from HFRS patients or rodents in Gyeonggi Province. Gangwon Province is a HFRS-endemic area with a high incidence of patients and prevalence of infected rodents, ROK. However, the continued epidemiology and surveillance of orthohantavirus remain to be investigated. Methodology/Principal findings Whole-genome sequencing of HTNV was accomplished in small mammals collected in Gangwon Province during 2015–2018 by multiplex PCR-based next-generation sequencing. To elucidate the geographic distribution and molecular diversity of viruses, we conducted phylogenetic analyses of HTNV tripartite genomes. We inferred the hybrid zone using cline analysis to estimate the geographic contact between two different HTNV lineages in the ROK. The graph incompatibility based reassortment finder performed reassortment analysis. A total of 12 HTNV genome sequences were completely obtained from A . agrarius newly collected in Gangwon Province. The phylogenetic and cline analyses demonstrated the genetic diversity and hybrid zone of HTNV in the ROK. Genetic exchange analysis suggested the possibility of reassortments in Cheorwon-gun, a highly HFRS-endemic area. Conclusions/Significance The prevalence and distribution of HTNV in HFRS-endemic areas of Gangwon Province enhanced the phylogeographic map for orthohantavirus outbreak monitoring in ROK. This study revealed the hybrid zone reflecting the genetic diversity and evolutionary dynamics of HTNV circulating in Gangwon Province. The results arise awareness of rodent-borne orthohantavirus diseases for physicians in the endemic area of ROK.

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

“…In the ROK, approximately 400 HFRS cases are reported annually, with a mean case fatality rate of 1–4% [ 33 ]. HFRS is highly endemic in Gyeonggi and Gangwon Provinces, ROK, and affects both military personnel and civilians [ 29 , 34 , 35 , 36 ]. Phylogenetic analyses of hantaviruses delineated a clear molecular epidemiological relationship between HFRS patients and their reservoir hosts, indicating the most putative site of infection [ 37 ].…”

“…cDNA was synthesized from 1 µg of total RNA by using the High Capacity RNA-to-cDNA kit (Applied Biosystems, Foster City, CA, USA) with a random hexamer and OSM55 (5′-TAG TAG ACT CC-3′). PCR conditions and primer sequences for amplification of HTNV genomes have been previously described [ 29 ]. Oligonucleotide primer sequences followed up Han-L-F1 (outer): 5′-ATG TAY GTB AGT GCW GAT GC-3′ and Han-L-R1 (outer): 5′-AAC CAD TCW GTY CCR TCA TC-3′, Han-L-F2 (inner): 5′-TGC WGA TGC HAC IAA RTG GTC-3′ and Han-L-R2 (inner): 5′-GCR TCR TCW GAR TGR TGD GCA A-3′ for the L segment; G2F1 (outer): 5′-TGG GTG CAA GTG C-3′, G2-2 (outer): 5′-ACA TGC TGT ACA GCC TGT GCC-3′, G2-1 (inner): 5′-TGG GCT GCA AGT GCA TCA GAG-3′, G2-4 (inner): 5′-ATG GAT TAC AAC CCC AGC TCG-3′ for the M segment; HTN-S6 (outer): 5′-AGC TCI GGA TCC ATI TCA TC-3′ and OSQ84 (outer): 5′-ATC TTA CAT CCT TTG TCG TCC C-3′, HTN-S4 (inner): 5′-GAI IGI TGT CCA CCA ACA TG-3′ and OSQ85 (inner): 5′-AGT TGT CCA CAG CCT CCT TT-3′ for the S segment.…”

A novel genotype of Hantaan orthohantavirus harbored by Apodemus agrarius chejuensis as a potential etiologic agent of hemorrhagic fever with renal syndrome in Republic of Korea

“…cDNA was synthesized from 1 μg of total RNA using the High Capacity RNA-to-cDNA kit (Applied Biosystems; Foster City, CA, USA) with OSM55 (5′-TAG TAG TAG ACT CC-3′). The PCR conditions were previously described [ 18 ]. All oligonucleotide primer sequences used in this study are shown in S6 Table .…”

“…The composition consisted of 12.5 μL 2× Uh pre-mix, 2.0 μL each primer mixture, 10 μL cDNA template, and 10.5 μL D.W. in 25 μL reaction mixture. The PCR cycling conditions were previously described [ 18 ]. The PCR amplicons were prepared using the TruSeq Nano DNA LT sample preparation kit (Illumina; San Diego, USA) according to the manufacturer’s instructions and previously described study [ 60 ].…”

“…agrarius collected in endemic regions [ 14 ]. However, most epidemiological surveillance studies have been performed in the northern ROK, which limits the understanding of the correlation between HFRS clinical cases and their reservoirs in southern areas [ 15 – 18 ]. Although a few studies have demonstrated the prevalence of orthohantavirus in the southern ROK, epidemiological investigations of orthohantavirus outbreaks in humans and rodents in the southern ROK were limited due to the lack of available viral sequences [ 19 – 23 ].…”