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

Kyungmin Park; Won-Keun Kim; Seung-Ho Lee; Jongwoo Kim; Jingyeong Lee; Seungchan Cho; Geum-Young Lee; Jin Sun No; Keun Hwa Lee; Jin-Won Song

doi:10.1371/journal.pntd.0009400

. 2021 May 12;15(5):e0009400. doi: 10.1371/journal.pntd.0009400

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

Kyungmin Park ^1,^2,^#, Won-Keun Kim ^3,^4,^#, Seung-Ho Lee ², Jongwoo Kim ², Jingyeong Lee ², Seungchan Cho ², Geum-Young Lee ², Jin Sun No ^2,⁵, Keun Hwa Lee ⁶, Jin-Won Song ^1,^2,^*

Editor: Brett M Forshey⁷

PMCID: PMC8143423 PMID: 33979351

Abstract

Background

Orthohantaviruses, causing hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome, pose a significant public health threat worldwide. Despite the significant mortality and morbidity, effective antiviral therapeutics for orthohantavirus infections are currently unavailable. This study aimed to investigate the prevalence of HFRS-associated orthohantaviruses and identify the etiological agent of orthohantavirus outbreaks in southern Republic of Korea (ROK).

Methodology/Principal findings

We collected small mammals on Jeju Island during 2018–2020. We detected the Hantaan virus (HTNV)-specific antibodies and RNA using an indirect immunofluorescence assay test and reverse transcription-polymerase chain reaction on Apodemus agrarius chejuensis (A. chejuensis). The prevalence of anti-HTNV antibodies among rodents was 14.1%. A total of six seropositive mouse harbored HTNV RNA. The amplicon-based next-generation sequencing provided nearly full-length tripartite genomic sequences of six HTNV harbored by A. chejuensis. Phylogenetic and tanglegram analyses were conducted for inferring evolutionary relationships between orthohantaviruses with their reservoir hosts. Phylogenetic analysis showed a novel distinct HTNV genotype. The detected HTNV genomic sequences were phylogenetically related to a viral sequence derived from HFRS patient in southern ROK. Tanglegram analysis demonstrated the segregation of HTNV genotypes corresponding to Apodemus spp. divergence.

Conclusions/Significance

Our results suggest that A. chejuensis-borne HTNV may be a potential etiological agent of HFRS in southern ROK. Ancestral HTNV may infect A. chejuensis prior to geological isolation between the Korean peninsula and Jeju Island, supporting the co-evolution of orthohantaviruses and rodents. This study arises awareness among physicians for HFRS outbreaks in southern ROK.

Author summary

According to the Korea Disease Control and Prevention Agency, about 18 HFRS cases have been reported over the past decade on Jeju Island, Republic of Korea (ROK). Currently, no report for the etiological agent associated with hemorrhagic fever with renal syndrome (HFRS) on Jeju Island is available. We report the nearly whole-genome sequences and have characterized a novel HTNV genotype carried by the Jeju striped field mice (Apodemus chejuensis) collected from Jeju Island. The phylogenetic analysis of HTNV revealed well-supported phylogeographic clusters, showing a high genetic divergence by the distribution of rodent reservoir hosts. To our knowledge, this study is the first provisional report that suggests molecular evidence of orthohantavirus outbreaks associated with hantaviral disease on Jeju Island. Although our observations are provisional, the novel genotype of HTNV harbored by A. chejuensis may be a potential etiological agent of HFRS in southern ROK. This study provides important insights into the male-predominance prevalence, genetic diversity, and molecular evolutionary dynamics of orthohantaviruses circulating in the ROK.

Introduction

Orthohantavirus (Family Hantaviridae, Order Bunyavirales) is an enveloped negative-sense single-stranded RNA virus containing large (L), medium (M), and small (S) genome segments [1]. The tripartite segments encode an RNA-dependent RNA polymerase (RdRp), two membrane glycoproteins (G_N and G_C), and a nucleocapsid (N) protein. Orthohantaviruses are etiological agents of hemorrhagic fever with renal syndrome (HFRS) with fatality rates of 1–15% in Eurasia [2]. Natural reservoirs of orthohantaviruses belong to various small mammals, including rodents (Rodentia), bats (Chiroptera), and insectivores (Soricomorpha) [3–5]. HFRS is mainly caused by Hantaan virus (HTNV), Seoul virus (SEOV), Dobrava virus (DOBV), and Puumala virus [6]. Orthohantaviruses are transmitted to humans by inhaling aerosolized infectious particles derived from saliva, urine, and feces of infected rodents. Antiviral therapeutics for orthohantavirus infections remain ineffective and unavailable despite the significant mortality and morbidity of the diseases.

Emerging orthohantaviruses pose a significant public health threat worldwide. According to the Korea Disease Control and Prevention Agency, approximately 400 HFRS cases are recorded annually in the Republic of Korea (ROK), with a mean mortality rate of 1–4% [7]. In 1976, HTNV harbored by the striped field mice (Apodemus agrarius), was first identified in the ROK [8]. In addition, five types of hantaviruses have been characterized: SEOV carried by Rattus norvegicus and R. rattus; Soochong virus (SOOV) carried by A. peninsulae; Muju virus carried by Myodes regulus; Imjin virus carried by Crocidura lasiura, and Jeju virus carried by C. shantungensis [9–13]. Previous studies have reported genomic and phylogeographic relationships between the patients with HFRS and A. 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]. On Jeju Island, an administrative island in southern ROK, approximately 18 HFRS cases have been reported from 2011 to 2019 [7]. Based on the incidence of HFRS, one or more distinct, rodent-borne orthohantaviruses have been suspected to exist on the island. However, the etiological agent of HFRS on Jeju Island still remains unknown.

In this study, we report the nearly full-length genome sequences and characterize a genetically distinct orthohantavirus, harbored by the subspecies A. agrarius chejuensis (A. chejuensis) captured on Jeju Island. The phylogenetic association of A. chejuensis-borne HTNV with viral sequences from HFRS patient suggests that A. chejuensis-borne HTNV may be a potential etiological agent of HFRS on Jeju Island, ROK. This study provides significant insights into the genetic diversity and molecular evolution of HTNV in the ROK. We highlight the necessity of continued surveillance and large-scale investigations for understanding the evolutionary diversification, transmission, and pathogenicity of orthohantaviruses in the southern areas, ROK.

Results

Trapping for small mammals on Jeju Island, ROK

Rodents and shrews were captured on Jeju Island, ROK from 2018 to 2020 (Fig 1). The trapped small mammals consisted of 91.4% (64/70) A. chejuensis, 5.7% (4/70) C. shantungensis, and 2.9% (2/70) Tscherskia triton (Table 1). A. chejuensis represented 91.4% of all individuals trapped on Jeju Island, and the most frequently captured species at all trapping sites. A. chejuensis captured on Jeju Island was genetically distinct from A. agrarius captured in mainland, ROK (S1 Fig).

Table 1. Trapping results of small mammals collected at sites on Jeju Island in the Republic of Korea, 2018–2020.

Species	Ara-dong	Aewol-eup	Bongseong-ri	Haengwon-ri	Ora-dong	Yongsu-ri	Total (%)
Apodemus chejuensis	12	5	7	19	15	6	64 (91.4)
Crocidura shantungensis	-	-	-	2	1	1	4 (5.7)
Tscherskia triton	-	-	-	2	-	-	2 (2.9)
Total	12	5	7	23	16	7	70 (100)

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Serological and molecular prevalence for HTNV collected on Jeju Island

A total of 9/64 (14.1%) rodents were seropositive, of which 1/12 (8.3%), 1/5 (20%), 3/7 (42.9%), 1/19 (5.3%), and 3/15 (20%) were detected in Ara-dong, Aewol-eup, Bongseong-ri, Haengwon-ri, and Ora-dong, respectively, with no seropositive rodents being detected in Yongsu-ri (Table 2 and S2 Fig). The prevalence of anti-HTNV antibodies was 7/35 (20%) in males and 2/29 (6.9%) in females. HTNV-specific reverse transcription-polymerase chain reaction (RT-PCR) was conducted for viral RNA. In total, 6/9 (66.7%) seropositive A. chejuensis harbored HTNV RNA, consisting of 5/7 (71.4%) males and 1/2 (50%) females. The antibodies against orthohantaviruses were not detected in C. shantungensis and T. triton.

Table 2. Serological and molecular prevalence of Hantaan virus (HTNV) in Apodemus chejuensis captured on Jeju Island from 2018 to 2020.

Trapping site	Number of captured A. chejuensis	Seropositivity for anti-HTNV IgG (%)			HTNV RNA positivity (%)
Trapping site	Number of captured A. chejuensis	Male	Female	Total	Male	Female	Total
Ara-dong	12	1/5 (20)	0/7	1/12 (8.3)	0/1	-^a	0/1
Aewol-eup	5	1/3 (33.3)	0/2	1/5 (20)	0/1	-	0/1
Bongseong-ri	7	2/4 (50)	1/3 (33.3)	3/7 (42.9)	2/2 (100)	1/1 (100)	3/3 (100)
Haengwon-ri	19	0/12	1/7 (14.3)	1/19 (5.3)	-	0/1	0/1
Ora-dong	15	3/9 (33.3)	0/6	3/15 (20)	3/3 (100)	-	3/3 (100)
Yongsu-ri	2	0/2	0/4	0/6	-	-	-
Total	64	7/35 (20)	2/29 (6.9)	9/64 (14.1)	5/7 (71.4)	1/2 (50)	6/9 (66.7)

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^a; Seronegative sample was not analyzed by RT-PCR

HTNV RNA loads in tissues from A. chejuensis

The HTNV RNA load in A. chejuensis was determined by quantitative PCR (qPCR) using various samples from the brains, lungs, livers, kidneys, spleens, reproductive organs, and hearts (Fig 2). Both IFA⁺PCR⁻ and IFA⁻PCR⁻ groups showed that HTNV RNA was undetectable in all tissues. In IFA⁺PCR⁺ rodents, viral RNA was identified in multiple tissues from rodents (Ac19-6, Ac20-5, Ac20-6, Ac20-30, Ac20-31, and Ac20-32). The lung tissues harbored the highest amount of HTNV RNA (Ct values 21.8–31.5) in all evaluated rodents except Ac20-32. The brain tissues showed the presence of HTNV RNA in all rodents except Ac20-6. The rodents (Ac20-5, Ac20-31, and Ac20-32) contained the HTNV RNA in reproductive organs.

Fig 2 — Ct values were determined for HTNV RNA of the S segment in brain, lung, liver, kidney, spleen, reproductive organ, and heart tissues obtained from six rodents positive for HTNV IgG and HTNV RNA. Ct, cycle threshold; S, small; HTNV, Hantaan virus; IgG, immunoglobulin G.

Whole-genome sequencing of HTNV

Using multiplex PCR-based next-generation sequencing (NGS), the nearly whole-genome sequence of HTNV was obtained from the lung tissues of A. chejuensis captured on Jeju Island. Table 3 shows that 41.8–93.8%, 97.3–99.9%, and 99.3–99.7% coverages of the L, M, and S segments of six A. chejuensis borne-HTNV. The average number of viral reads and depth of coverage is shown in the S1 Table. The uncovered sequences of HTNV tripartite RNA were determined from HTNV Ac19-6, Ac20-5, Ac20-6, Ac20-30, Ac20-31, and Ac20-32, using conventional RT-PCR. To complete the whole-genome sequence of HTNV, 5′ and 3′ end rapid amplification of cDNA ends (RACE) PCR was performed in HTNV Ac20-30. The 5′ termini genome sequences of HTNV Ac20-30 were recovered in tripartite segments, while the 3′ end viral sequence was obtained only in the M segment. The 5′ end sequences of HTNV were 5′-GGA GUC UAC UAC UA-3′ in the tripartite segments. The 3′ termini sequence of the HTNV M segment was 5′-UAG UAG UAU GCU CC-3′, showing incomplete complementarity due to a mismatch at position 9 and a noncanonical U–G pair at position 10.

Table 3. Next-generation sequencing coverages of Hantaan virus (HTNV) from rodents collected in Jeju Island, 2018–2020.

Site	Sample	IFA for anti-HTNV IgG	Origin	Ct value	HTNV genomes, % coverage
Site	Sample	IFA for anti-HTNV IgG	Origin	Ct value	L segment	M segment	S segment
Ora-dong, Jeju-si	Ac19-6	1:1,024^a	Lung	30.0	81	96.6	98.1
	Ac20-5	1:32^b	Lung	23.8	58.6	99.2	98.4
	Ac20-6	1:16^b	Lung	31.5	41.4	97	98.4
Bongseong-ri, Jeju-si	Ac20-30	1:256^a	Lung	25.9	93.4	99.1	98.4
	Ac20-31	1:256^a	Lung	21.8	93	99	98.4
	Ac20-32	1:8^b	Lung	27.7	91	99.1	98.4

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Ac, Apodemus chejuensis; IFA, indirect immunofluorescence antibody test; IgG, immunoglobulin G; C_t, cycle threshold; L, large; M, medium; S, small.

^a; IFA test was performed from sera

^b; IFA test was performed from heart fluids.

Genome coverage was calculated by obtaining consensus sequences matching the prototype HTNV 76–118.

The complete genome sequence lengths of HTNV are the L (6,533 nt), M (3,616 nt), and S segments (1,696 nt), respectively.

Genomic characterization of HTNV

The nucleotide identity of the HTNV L, M, and S segments from Jeju Island showed 82.2–83.1%, 87.2–88.8%, and 84.8–85.4%, respectively, compared to those of HTNV 76–118 (S2–S4 Tables). The amino acid sequences differed between HTNV from Jeju Island and HTNV 76–118 by 2.8–3.9%, 1.8–2.5%, and 3.3–3.7%. The entire M segment of HTNV harbored by A. chejuensis was 3,618 nucleotides with a glycoprotein precursor of 1,135 amino acids, with the highly conserved pentapeptide WAASA motif being found at amino acid positions 644–648. HTNV strains from A. chejuensis showed seven potential N-linked glycosylation sites in two glycoproteins: five in Gn (N134, N235, N347, N399, and N609) and two in Gc (N766 and N928) (S3 Fig). The glycosylation sites were identical to those of HTNV 76–118 harbored by A. agrarius.

Phylogenetic and co-evolutionary analyses of HTNV

The phylogenetic analysis of the HTNV L, M, and S segments showed well-supported geographical clusters (Fig 3). The L segment of HTNV from Jeju Island formed an independent geographic and distinct cluster with all other viral genomes collected from mainland ROK and China. The M segment of HTNV from Jeju Island shared a common ancestor with the HTNV from mainland ROK. The S segment of HTNV from Jeju Island formed an independent genetic lineage with other HTNV, SOOV, and Amur virus (AMRV). In addition, the phylogenetic analysis of the partial HTNV L, M, and S segments revealed well-supported geographical clusters (Fig 4). In all segments, A. chejuensis-borne HTNV from Jeju Island formed homologous genetic clades with CUH15-126 obtained from the patients with HFRS in Jangheung-gun (Jeollanam Province) and distinguished from other A. agrarius-borne HTNV (Gyeonggi and Gangwon Provinces). Tanglegram analysis showed the segregation of representative rodent-borne hantaviruses according to the species of their reservoir hosts (Fig 5). The phylogenetic positions of HTNV from A. chejuensis and other hantaviruses mirrored the co-evolutionary relationships of their Rodentia hosts, except for Tula and Lúxī virus.

Fig 4 — (A) Geographic locations (circle symbols) indicate where the HTNV genomes were acquired in mainland and Jeju Island, ROK. The circle indicates geographic sites (a, Jeju; b, Jangheung; c, Paju; d, Yeoncheon; e, Pocheon; f, Cheorwon; g, Yanggu). Phylogenesis were generated by maximum-likelihood based on the alignment (Clustal W method) using the (B) 353-nucleotide L (coordinates 2,963–3,315 nt), (C) 369-nucleotide M (coordinates 1,973–2,341 nt), and (D) 639-nucleotide S (coordinates 412–1,050 nt) segments of HTNV. Topologies were evaluated using bootstrap analysis of 1,000 iterations. The following viral sequences were included in the analysis: HTNV ROKA13-8 (M segment, KU207202; S segment, KU207206), ROKA14-11 (L segment, KU207199; M segment, KU207203; S segment, KU207207), ROKA16-9 (L segment, MH598466; M segment, MH598480; S segment, MH598494), ROKA17-3 (L segment, MH598467; M segment, MH598481; S segment, MH598495), ROKA17-5 (L segment, MH598468; M segment, MH598482; S segment, MH598496), ROKA17-7 (L segment, MH598469; M segment, MH598483; S segment, MH598497), ROKA17-8 (L segment, MH598470; M segment, MH598484; S segment, MH598498), US8A14-2 (S segment, KU207208), US8A15-1 (L segment, KU207201), CUH15-126 (L segment, MG663537; M segment, MG663538; S segment, MG663539), Aa04-722 (L segment, KU2071740), Aa09-410 (L segment, KU207177), Aa09-948 (L segment, KT934966), Aa14-188 (M segment, KT935009), Aa15-56 (M segment, KU207187; S segment, KU207195), Aa15-58 (M segment, KU207188; S segment, KU207196), Aa17-7 (L segment, MH598475; M segment, MH598489; S segment, MH598503), Aa17-8 (L segment, MH598476; M segment, MH598490; S segment, MH598504), Aa17-48 (L segment, MH598477; M segment, MH598491; S segment, MH598505), Aa17-53 (L segment, MH598479; M segment, MH598493; S segment, MH598507), Aa18-179 (L segment, MT012580; M segment, MT012568; S segment, MT012556), Aa18-185 (L segment, MT012581; M segment, MT012569; S segment, MT012557), Ac19-6 (L segment, MW219756; M segment, MW219762; S segment, MW219768), Ac20-5 (L segment, MW219757; M segment, MW219763; S segment, MW219769), Ac20-6 (L segment, MW219758; M segment, MW219764; S segment, MW219770), Ac20-30 (L segment, MW219759; M segment, MW219765; S segment, MW219771), Ac20-31 (L segment, MW219760; M segment, MW219766; S segment, MW219772), Ac20-32 (L segment, MW219761; M segment, MW219767; S segment, MW219773), 76–118 (L segment, NC_005222; M segment, M14627; S segment, M14626), and SEOV 80–39 (L segment, NC_005238; M segment, NC_005237; S segment, NC_005236). Abbreviations; ROKA, Republic of Korea Army; US8A, United States 8^th Army forces; CUH, Chosun University Hospital; Aa, *Apodemus agrarius*; Ac, A. *chejuensis*; SEOV, Seoul virus.

Fig 5 — Tanglegram was generated using the R package, using consensus maximum likelihood topologies based on the nucleotide sequences of the hantaviral M segment (right panel) with mitochondrial DNA cytochrome b gene sequences of the reservoir host species (left panel). Letters for taxa are shown in red for *Murinae*, green for *Arvicolinae*, blue for *Signodontinae*, orange for *Neotominae*, black for *Soricidae* in left panel. *Apodemus chejuensis*-borne HTNV (Ac20-5) is shown in bold lettering in each panel. The host species and viruses accession number followed up; reservoir hosts *Crocidura shantungensis* (HQ993053), A. *agrarius* (MN205319), A. *chejuensis* (MW219775), A. *peninsulae* (AB073811), A. *flavicollis* (JF819967), *Hylomyscus simus* (DQ212188), *Rattus norvegicus* (AB355903), *Myodes regulus* (NC_016427), *Myodes glareolus* (AF119272), *Eothenomys miletus* (AY426686), *Microtus arvalis* (EU439459), *Peromyscus maniculatus* (AF119261), *Oligoryzomys longicaudatus* (AF346566), hantavirus strains JJUV 10–11 (NC_034404), HTNV 76–118 (M14627), HTNV Ac20-5 (MW219763), SOOV SC-1 (AY675353), DOBV Af19 (NC_005234), SANGV Sa14 (NC_034516), SEOV 80–39 (NC_005237), MUJV 11–1 (JX028272), PUUV Sotkamo (HE801634), TULV 5302v (NC_005228), LUXV LX3009 (NC_038528), SNV NMH10 (NC_005215), ANDV CHI7913 (AY228238). JJUV, Jeju virus; HTNV, Hantaan virus; SOOV, Soochong virus; DOBV, Dobrava virus; SANGV, Sangassou virus; SEOV, Seoul virus; MUJV, Muju virus; PUUV, Puumala virus; TULV, Tula virus; LUXV, Lúxī virus; SNV, Sin Nombre virus; ANDV, Andes virus.

Genetic reassortment analysis of HTNV

Recombination Detection Program 4 (RDP4) and Graph incompatibility based Reassortment Finder (GiRaF) analyses were performed to evaluate the possibility of genetic reassortment events. The genetic exchange event was undetectable among HTNV genomic sequences in this study.

Discussion

Hantaviruses have specifically evolved along with one or a few closely related rodent species [4,5,24]. The genus Apodemus (family Muridae, order Rodentia) consists of at least 20 small rodent species and is mainly widespread in the Palearctic area [25]. Molecular data based on the rodent mitochondrial DNA cytochrome b gene and the interphotoreceptor retinoid binding protein gene in nuclear DNA revealed that Apodemus spp. diverged approximately 8–10 million years ago [26]. In Asia, A. agrarius was first identified as the primary rodent reservoir of HTNV [8]. A. peninsulae was also a rodent reservoir for SOOV (closely related to AMRV) distributed in ROK, China, and Russia [10,27,28]. The striped field mouse (A. agrarius coreae), the most abundant rodent species, are widely distributed in the Korean peninsula, whereas A. agrarius chejuensis is mainly found on Jeju Island, the southernmost island of ROK [29]. However, a population of A. agrarius that are genetically and morphologically clustered with A. chejuensis has been reported in Wan Island (80 km North from Jeju Island) located on the southern coast of ROK [30]. Jeju Island was formed by a series of volcanic activities about 1.2 million years ago [31–34]. The island was connected to the Korean peninsula during the Pleistocene and separated about 10,000 years ago. The ancestral population of A. chejuensis on Jeju Island may have originated from the Korean peninsula or eastern China [35]. Han et al. and Koh et al. estimated the population of A. agrarius and A. chejuensis diverged approximately 12 million years ago and 7,000–500,000 years ago (Pleistocene period), respectively, although both studies referred to the last ice age as a crucial period [30,36]. A. agrarius and A. chejuensis have recently been suggested to be separate genera of Apodemus spp. based on mitochondrial genome analysis, morphological characteristics, and crossbreeding experiments [30,36,37]. The present study proposes that A. chejuensis-borne HTNV is a genetically distinct genotype due to the clear natural host species classification and co-divergent evolution of HTNV and Apodemus spp. after the geographic segregation between Jeju Island and the Korean peninsula. The species demarcation criterion of the International Committee for Taxonomy of Viruses (ICTV) is currently a 7% difference in amino acid sequences of G_N/G_C and N proteins. The differences in amino acid sequences of the RdRp, glycoproteins, and N proteins of HTNV strains harbored by A. chejuensis and A. agrarius did not exceed 5%. Taken together, A. chejuensis-harbored HTNV is considered Hantaan orthohantavirus species, including A. agrarius-borne HTNV and A. peninsulae-borne SOOV (closely related to AMRV). Continued surveillance and large-scale investigations should be conducted to understand the evolutionary diversification and geographic distribution of orthohantavirus and their natural reservoir hosts in ROK.

Rodent-borne orthohantaviruses pose a critical public health threat worldwide. In the ROK, approximately 400 HFRS cases are reported annually, with 33 mortality cases over a decade [7]. Approximately 70% HFRS cases are caused by A. agrarius-borne HTNV and the remaining by SEOV carried by R. norvegicus or other unidentified agents. Jeju Island includes urban and rural regions with a population of approximately 700,000. It is a popular tourism destination for international tourists to be listed as a Biosphere Reserve (2002), World Natural Heritage Site (2007), and Global Geopark (2010). Overall, visitation to the site has been sustainably increased by an influx of international tourists due to global reputations [38]. The island has been an endemic HFRS region since the first clinical case was officially identified in 2005. After five HFRS cases were recorded from 2001 to 2009, 18 clinical cases have been reported in the recent decade on Jeju Island [7]. Despite the distribution of the rodent reservoir population, HTNV and SEOV have not yet been detected on Jeju Island. In the present study, the genetically distinct HTNV was detected in A. chejuensis captured on Jeju Island. Phylogenetic analyses of orthohantaviruses delineated a clear molecular epidemiological link between the patients with HFRS and their reservoir hosts collected at the infection sites [39]. In particular, active surveillance with targeted rodent trapping in the sites suspected of orthohantavirus outbreaks associated with HFRS allowed for phylogenetic distinction within a relatively short distance (about 5 km) to track the precise infectious agents and potential exposed sites [14]. Our previous studies demonstrated a phylogenetic relationship of partial HTNV M segment genome between four US soldiers diagnosed with HFRS and HTNV-positive A. agrarius collected at the very likely infection sites [40]. The phylogenetic analysis showed that HTNV derived from A. chejuensis genetically linked CUH15-126, which is the only hantaviral sequence collected from the patient with HFRS in southern ROK (Jeollanam Province) [41]. These results indicate that A. chejugensis-borne HTNV may be a potential etiological agent in clinical HFRS cases in southern ROK.

The subdivision of DOBV genotypes (Dobrava, Kurkino, Saaremaa, and Sochi) was differentiated by the characteristics in their phylogeny, rodent reservoirs, and geographical distribution [42]. Despite their high genetic homology, different DOBV genotypes induce HFRS with varied severities. The case-fatality rate (CFR) of clinical cases due to the Dobrava genotype harbored by A. flavicollis was 10–12% [43–45]. The CFR of HFRS cases caused by the Sochi genotype harbored by A. ponticus was approximately 6% [46]. Furthermore, the CFR range for orthohantavirus outbreaks associated with the Kurkino genotype carried by A. agrarius was 0.3–0.9% in European Russia. A. agrarius-borne SAAV genotype infection appears to be subclinical [47,48]. Thus, the transmission, pathogenicity, and disease risk assessment of HFRS by distinct A. chejugensis-borne HTNV in humans should be determined.

Genomic reassortment plays an important role in the evolutionary mechanism by which segmented RNA viruses may shuffle genome segments to generate new viral strains [49]. Genetic exchange of hantaviruses occurs more frequently between genetically closed strains than between distant strains. The incongruence of phylogenetic patterns in the tripartite segments showed the possibility of genetic reassortment among hantaviruses [46,50]. In this study, the phylogenies of A. chejuensis-borne HTNV L, M, and S segments revealed differing levels of incongruence in phylogenetic positions, which indicates the differential evolution of each segment. The L segment of the HTNV shared common ancestors with other HTNV lineages collected from mainland ROK and China. The M segment formed a clade with HTNV strains in the ROK. The S segment showed the independent geographic and distinct cluster with Hantaan orthohantavirus, including HTNV, SOOV, and AMRV. The phylogenetic pattern of A. chejuensis-borne HTNV may be a configuration compatible with genetic reassortment. However, recombination and reassortment were considered insignificant by RDP4 and GiRaF, respectively. To better understand the complexity of orthohantavirus evolution, future studies should focus on continuous sample collection from reservoir hosts and analysis of hantaviral sequences.

Sexual maturity and aggression are associated with viral transmission among natural host reservoirs [51,52]. The prevalence of orthohantaviruses in rodent populations plays a critical role in understanding hantaviral disease in HFRS-endemic areas. The seroprevalence of orthohantaviruses in rodents demonstrated a higher prevalence of infection in males than females [16,53–57]. Hinson et al. reported that wounded male rats were more frequently infected with SEOV than non-wounded male rats [58]. In this study, the prevalence of HTNV antibodies and RNA was higher in males (7/35, 20%; 5/7, 71.4%, respectively) than those in females (2/29, 6.9%; 1/2, 50%, respectively). Our results showed a gender-specific prevalence of HTNV-infected A. chejuensis, supporting that males may contribute to the spread of viral infection via competition with other rodents. However, the number of samples (n = 64) was limited in this study. Thus, continuous large-scale epidemiological surveillance should be conducted to clarify the pattern of male-preferential prevalence of HTNV-infected A. chejuensis.

In conclusion, a genetically distinct HTNV was found in A. chejuensis captured on Jeju Island, ROK. The phylogeny of orthohantaviruses and their rodent hosts demonstrated that A. chejuensis may be infected with ancestral HTNV prior to geological isolation. Our results suggest that A. chejuensis-borne HTNV may be a potential etiological agent of HFRS in southern ROK. The discovery of a novel HTNV genotype provides important insights into the evolutionary history, genetic diversity, gender-specific prevalence of orthohantaviruses and a clue to account for HFRS cases that cannot be attributed to A. agrarius-borne HTNV infection in the ROK.

Methods

Ethics statement

Small mammals were handled in accordance with the Ethical Guidelines of the Korea University Institutional Animal Care and Use Committee (KUIACUC #2019-4 and 2019-171). Captured rodents and shrews were euthanized via cardiac puncture under alfaxalone-xylazine anesthesia. The experiment was performed in an animal biosafety level 3 facility at Korea University.

Sample collection

Small mammals were captured using Sherman live traps (8 × 9 × 23 cm; H. B. Sherman; Tallahassee, FL, USA) on Jeju Island, ROK from 2018 to 2020. A total of 100 traps were placed in unmanaged grasses, herbaceous vegetation, farmlands, mountains, fields, and forests for 1–3 days (S5 Table). The captured small mammals were identified morphologically and transported to Korea University in accordance with the safety guidelines. Multiple tissues of the rodents and shrews were dissected aseptically and the sera were separated by centrifugation for 5 min at 4°C. The tissue samples were stored at −80°C until use.

Mitochondrial DNA (mtDNA) analysis

Total DNA was extracted from liver tissues using a High Pure PCR Template Preparation Kit (Roche; Basel, Switzerland). The cytochrome b gene of mtDNA was amplified using universal primers [59]. The sequences were deposited in GenBank (accession numbers: MW219774- MW2197749).

Indirect immunofluorescence antibody (IFA) test

The sera and heart fluids of small mammals were used to detect anti-HTNV immunoglobulin G (IgG). The sera or heart fluids were diluted 1:32 and 1:2, respectively, in phosphate buffered saline (PBS) and added to acetone-fixed Vero E6 cells infected with HTNV. The slide was incubated at 37°C for 30 min. The cells were washed with PBS and distilled water (D.W.). The slides were treated with fluorescein isothiocyanate-conjugated anti-mouse (for A. chejuensis and T. triton) or mouse/rat (for C. shantungensis) IgG (ICN Pharmaceuticals; Laval, Quebec, Canada). The plates were incubated at 37°C for 30 min and washed with PBS and D.W. The virus-specific fluorescence was evaluated using a fluorescent microscope (Axio Scope; Zeiss; Berlin, Germany).

Reverse transcription-polymerase chain reaction (RT-PCR)

Total RNA was extracted from lung tissues of rodents and shrews with TRI Reagent Solution (Ambion; Austin, Texas, USA). 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.

Quantitative PCR (qPCR)

qPCR was performed in a 10 μL reaction mixture containing 1 μg of total RNA. The cycling conditions consisted of denaturation at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s, and 60°C for 1 min using SYBR Green PCR Master Mix (Applied Biosystems) on a QuantStudio 5 Real-Time PCR System (Applied Biosystems).

Multiplex PCR-based next-generation sequencing (NGS)

cDNA was amplified using HTNV-specific primer mixtures and Solg 2X Uh-Taq PCR Smart mix (Solgent; Seoul, Republic of Korea) according to the manufacturer’s instructions. 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]. NGS was performed on a MiSeq benchtop sequencer (Illumina) with 2 × 150 bp using a MiSeq reagent kit v2 (Illumina). The output files were imported and analyzed by CLC Genomics Workbench version 7.5.2 (CLC Bio; Cambridge, MA, USA). The sequences of HTNV strains were deposited in GenBank (accession numbers: MW219756- MW219773).

Rapid amplification of cDNA ends (RACE) PCR

To obtain the termini 3′ and 5′ genome sequences of HTNV, RACE PCR was conducted using a 3′- and 5′- RACE System for Rapid Amplification of cDNA Ends, Version 2.0 (Invitrogen; Carlsbad, CA, USA), according to the manufacturer’s specifications.

Phylogenetic analysis

The tripartite genomic sequences of HTNV were aligned using the Clustal W algorithm in Lasergene version 5 (DNASTAR Inc.; Madison, WI, USA). For nearly full-length HTNV, phylogenetic analysis was performed using the best-fit GTR+G+I (for L and M segments) and GTR+G (for S segment) substitution models of evolution by the maximum-likelihood (ML) method in MEGA 7. Partial phylogenetic trees were generated by the ML method under the optimal evolutionary models T92+I (for L segment) and T92+G (for M and S segments). The topologies were assessed by bootstrap analysis for 1,000 iterations.

Co-divergence analysis

A tanglegram algorithm was generated to compare different phylogenetic links matching between HTNV strains and their hosts. The auxiliary lines in the center connect between the phylogenetic trees. The complexity between dendrograms of phylogenies was reduced to the maximum before the full reconciliation analysis. The method was implemented in the R package (dendextend) [61].

Genetic reassortment analysis

Alignment analysis of the concatenated HTNV L, M, and S segment open reading frame regions were performed using RDP, GENECONV, MAXCHI, CHIMAERA, 3SEQ, BOOTSCAN, and SISCAN in the RDP4 software [62]. A p-value of recombination and reassortment events under 0.05 was considered statistically significant. Genetic events were likely to indicate the occurrence of a genetic exchange when p-value was under 0.05, and the RDP recombination consensus score (RDPRCS) was between 0.4 and 0.6. All parameters of RDP4 were left at the default.

The GiRaF was performed to confirm reassortment events [63]. Nucleotide alignments of the tripartite segments were used as an input source for MrBayes [64]. The best nucleotide substitution models were determined by using MEGA 7. As input for the software, 1,000 unrooted candidate trees were estimated using the GTR+G+I substitution model, the burn-in 50,000 iterations (25%), and sampling every 200 iterations. These trees were used to simulate the phylogenetic uncertainty for segments; the parameters of the GiRaF were default settings. The process was repeated ten times, with ten independent MrBayes based tree data per segment.

N-glycosylation site analysis

For N-glycosylation site prediction, the full-length amino acid sequences were submitted to the NetNGlyc 1.0 server (https://services.healthtech.dtu.dk) and the parameter was set to default [65].

Supporting information

S1 Fig. A phylogenetic tree based on the mitochondrial DNA cytochrome b gene of Apodemus spp.

Jeju striped field mice (A. chejuensis) from Jeju Island were confirmed by conventional polymerase chain reaction (PCR) for the mitochondrial DNA cytochrome b gene (coordinates 119–1,080 nt). The phylogenetic tree was generated by the maximum likelihood method using MEGA 7.0. A bootstrap support value >70% is described at the nodes. Apodemus mice are color-coded according to geographic regions (red, mainland ROK; blue, Jeju Island of ROK; green, China).

(TIF)

Click here for additional data file.^{(5.3MB, tif)}

S2 Fig. Detection of immunoglobulin G (IgG) antibodies against Hantaan virus (HTNV) from Apodemus chejuensis sera and heart fluids by indirect immunofluorescence assay (IFA).

HTNV-infected Vero E6 cells were fixed on each slide. The sera (1:32 dilution) or heart fluids (1:2 dilution) of rodents were used to detect anti-HTNV IgG by IFA, Ac18-6, Ac18-17, Ac19-6, Ac20-30, and Ac20-31; heart fluids of Ac20-5 and Ac20-6. Positive and negative controls used were serum (Aa18-185) and phosphate buffered saline (PBS), respectively. Ac, A. chejuensis; Aa, A. agrarius.

(TIF)

Click here for additional data file.^{(4.2MB, tif)}

S3 Fig. Prediction of N-glycosylation sites in full-length glycoproteins of HTNV and other representative hantaviruses.

The N-linked glycosylation sites for the entire length of glycoproteins in hantaviruses were predicted using NetNGlyc 1.0 server (DTU Bioinformatics), and the parameter was set to default. The potential N-linked glycosylation sites for rodent-borne orthohantaviruses (HTNV, SOOV, and SEOV) are shown. HTNV, Hantaan virus; SOOV, Soochong virus; SEOV, Seoul virus.

(TIF)

Click here for additional data file.^{(4.1MB, tif)}

S1 Table. Summary of mapped reads and depth of coverages by Hantaan virus (HTNV) multiplex PCR-based next-generation sequencing.

(PDF)

Click here for additional data file.^{(107.1KB, pdf)}

S2 Table. Percentage similarity based on L segment of HTNV nucleotide and amino acid sequences between HTNV from Jeju Island and representative rodent-borne orthohantaviruses.

(PDF)

Click here for additional data file.^{(100.7KB, pdf)}

S3 Table. Percentage similarity based on M segment of HTNV nucleotide and amino acid sequences between HTNV from Jeju Island and representative rodent-borne orthohantaviruses.

(PDF)

Click here for additional data file.^{(111.8KB, pdf)}

S4 Table. Percentage similarity based on S segment of HTNV nucleotide and amino acid sequences between HTNV from Jeju Island and representative rodent-borne orthohantaviruses.

(PDF)

Click here for additional data file.^{(111.4KB, pdf)}

S5 Table. Topography and GPS coordinates of the trapping sites.

(PDF)

Click here for additional data file.^{(70.2KB, pdf)}

S6 Table. Oligonucleotide primer sequences generated in this study.

(PDF)

Click here for additional data file.^{(120.4KB, pdf)}

Acknowledgments

We thank Dr. Man-Seong Park for supporting experiments.

Data Availability

The mitochondrial DNA sequences were deposited in GenBank (accession numbers: MW219774- MW2197749). The genomic sequences of HTNV were deposited in GenBank (accession numbers: MW219756- MW219773).

Funding Statement

This study was supported by the Research Program to Solve Social Issues of the National Research Foundation of Korea (NRF) funded by the Ministry of Science and Information and Communication Technology (ICT) (NRF-2017M3A9E4061992 to J.-W.S.) and the Agency for Defense Development (UE202026GD to J.-W.S.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS Negl Trop Dis. doi: 10.1371/journal.pntd.0009400.r001

Decision Letter 0

Brett M Forshey, Emma Wise

19 Mar 2021

Dear Dr. Song,

Thank you very much for submitting your manuscript "A novel genotype of Apodemus chejuensis-borne Hantaan Orthohantavirus as a potential etiologic agent of Hemorrhagic Fever with Renal Syndrome in Republic of Korea" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

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PLOS Neglected Tropical Diseases

***********************

Kyungmin Park and co-authors analyze the prevalence and diversity of hantaviruses in rodents on Jeju Island, identifying a HTNV in Apodemus chejuensis that is genetically distinct from other HTNV in other rodents. However, this HTNV was genetically related to a sequence from a patient from southern ROK, which suggests that A. chejuensis-associated HTNV might be the cause of HFRS. This is a well-written manuscript, and the analysis and interpretation is solid. The reviewers had mostly minor comments. In addition to the comments from the reviewers below, please consider the following additional minor suggestions:

Abstract, line 37: "The HTNV genomic sequences were phylogenetically related to a viral sequence from the patients with HFRS in southern ROK" - wouldn't this be more precise to say "patient" instead of "patients" since there was only one sequence available?

Abstract, lines 89-90: "We also highlight preemptive precautions for preventing and controlling hantaviral outbreaks to physicians on Jeju Island" I don't recall seeing much in the way of preemptive precautions in the manuscript - this may need to be drawn out a bit more in a couple of sentences in the Discussion.

Results, lines 121-122: "Both IFA+PCR− and IFA−PCR− groups showed that the Ct value was 40, indicating that HTNV RNA was not detectable in tissues." Was the Ct value 40, or not detectable? If HTNV RNA was not detectable, then the Ct value wouldn't be 40, more likely there would be no Ct value to mention. Please clarify here and on Figure 2.

Results, lines 149-150: "The nucleotide similarity of the HTNV L, M, and S segments from Jeju Island compared to those of HTNV 76118 showed 82.2–83.1%, 87.2–88.8%, and 84.8–85.4% homology, respectively" Please check on the use of the terms "similarity" and "homology" in this sentence - would "identity" be more appropriate for nucleotide comparisons?

Discussion, lines 263-264: "Taken together, the distinct genotype of A. chejuensis-harbored HTNV is considered a single species of A. agrarius-borne HTNV" The use of genotype and species in this sentence seem at odds with one another - it's not clear that "species" is the right use of the word in this context. On a similar note, please see the comment from a reviewer below about the use of the term "strain" - consider whether "sequence" or some other term might be more technically precise.

Discussion, lines 274-275: So, from the 18 clinical HFRS cases on Jeju Island over the past decade, there were no sequences available for comparison?

Discussion, lines 305-308: "The phylogenetic pattern of A. chejuensis-borne HTNV may be a configuration compatible with genetic reassortment. However, recombination and reassortment were considered insignificant by Recombination Detection Program 4 and Graph-incompatibility-based Reassortment Finder, respectively (data not shown)." I think it would be useful to include these tools in the Methods, particularly for the "reassortment finder" program, since it gave a result that is not obvious from the phylogenetic tree.

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: YES, the study and it's design was appropriate to support the findings.

Reviewer #2: -Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

Almost yes.

-Is the study design appropriate to address the stated objectives?

Yes,

-Is the population clearly described and appropriate for the hypothesis being tested?

L 260: Species Demarcation criterion stated here seems to be outdated. Please check for more recent criterion in ICTV using concatenated amino acid sequences of N and Gn/Gc proteins of hantaviruses to demarcate the species. I agree novel sequences from Cheju islands were a novel "genotype" of Hantaan virus. But you should show results with new criteria.

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

Yes

-Were correct statistical analysis used to support conclusions?

Why did you ignore Niviventer borne-Hantaan viruses in this manuscript? I can see the relationship of novel genotype and Amur viruses. In phylogenetic analyses, please include Niviveter borne-viruses. You discuss about reassortment events. For this purpose, More Chinese Hantaan viruses should be included in phylogeny.

-Are there concerns about ethical or regulatory requirements being met?

Yes

Reviewer #3: see final comments.

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: The results and representations were correlatable with the objectives of the experiment but there are certain area which the author needs to clarify in order for better representation.

Reviewer #2: -Does the analysis presented match the analysis plan?

Yes

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #3: see final comments.

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: yes.

Reviewer #2: -Are the conclusions supported by the data presented?

Yes

-Are the limitations of analysis clearly described?

Yes

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

Yes

-Is public health relevance addressed?

It has been reported that mice derived from Cheju island were used to isolate prototype Hantaan virus strain 76118 virus by Dr. H. W. Lee (Hantavirus Hunting). At that time, they explained that this island was HFRS patient-free, so that they used apodemus mice derived from Jeju island. But you clearly showed existence of Hantaan virus in apodemus mice in this island. You have to discuss about following for public health problem?

Is there any Apodemus agrarius genotype in this island?

Is it possible that the Jeju-derived genotype is less pathogenic?

Since when has this virus been in this island? Host mouse was subspecies of A. agrariu. Rescently, it was considered independent species. Anyway, it was endemic only Cheju island. You have to clearly explain in explanation in Fig. 5

Reviewer #3: see final comments.

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: Minor revision

Reviewer #2: L 34: The sentence should better to be written as “The prevalence of anti-HTNV antibodies among rodents was 14.1%.”

L 36: The sentence should better to be written as “The detected HTNV genomic sequences were phylogenetically related to a viral sequence derived from HFRS patients in southern ROK.”

L 60: include the word “genome” before the word segments.

L 67: include “of infected rodents” after the word feces.

Figure 1: Is there any special reason to capture rodents only from Jeju-si part of the island?

Figure 2: Have you tried calculating the copy numbers of RNA in tissues?

Figure 4: The sequences for the phylogenetic analysis have been selected only from northern-most and southern-most regions of ROK. Is there a special reason not to select any sequence originated from central or other parts of the country?

Table 3:

1. Add “from Jeju island” at the end of the table title.

2. The term “strain” refers to the isolated and established virus. These are basically the sequences detected from rodent tissues. Therefore, it is preferred to use another term instead of “strain” in all the places (especially in abstract) stating it in the manuscript.

L 319: Is there an explanation for the low sample number even though the trapping was conducted for 2 years using relatively large number of traps?

L 348: Why low dilutions were used for IFA assay? The background reactions can be quite higher at such a low dilution level.

L 350/351: Why only a single secondary antibody was used to screen three species of rodent serum samples? Is there a cross reactivity to anti-mouse IgG in shrew and T. triton sera? In our experience the said secondary antibody does not react with shrew sera.

L 361: RT-qPCR primers were targeted at M-segment of HTNV. Is there any reason to choose M-segment over S-segment which has more copies in the virus for the quantification?

Reviewer #3: see final comments.

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: The study entitled “A novel genotype of Apodemus chejuensis-borne Hantaan Orthohantavirus as a potential etiologic agent of Hemorrhagic Fever with Renal Syndrome in Republic of Korea” appears to be a fascinating approach to identify the novel genotype as a pivotal etiologic agent of HFRS.

However, there are certain minor areas in which the author needs to clarify in order to refine the work for better representation.

1. Line no. 25 – Repetition of the word “pulmonary”. Hantavirus cardiopulmonary syndrome (HCP)

2. Line 34 - The expression of sentence is not very clear. What does the author mean to say, should clearly explain?

3. Line 87 - HFRS-Borne or Rodent-borne? HTNV is the causative agent of HFRS.

• What does the author mean to say by the phrase clinical HFRS-borne HTNV strain?

4. Line 95 – 64 should be replaced with sixty-four.

5. Line 108 - The author has mentioned about the collection of shrews and rodents both for the experiment. But the data related to shrews are missing.

• Was there any seropositive case for shrews as well?

6. Line 124 – What was the estimated cut-off value for the RT-qPCR. Author did not mention about that.

Reviewer #2: Kyungmin Park and colleagues have carried out an important study to reveal a potential HFRS causative agent. The study findings show interesting epidemiological features in the new-found HTNV sequences from A. chejuensis in Jeju island, ROK. Even though the total sample number, seropositivity and RNA detection rate is relatively low, the findings are satisfactory enough to justify the importance of studying the potential host rodent species that can harbor hantaviruses in HFRS endemic regions. The close phylogenetic relatedness of new-found HTNV sequences to the HFRS-patient derived HTNV sequences, is a critical observation that alarms the high possibility of this virus genotype to be a candidate etiological agent of HFRS in southern ROK. Overall study is well designed and well implemented. Methods used by the authors are suitable enough to yield significant results. The objectives of the study are well full filled by these findings.

Reviewer #3: The following review is for:

“A novel genotype of Apodemus chejuensis-borne Hantaan Orthohantavirus as a potential etiologic agent of Hemorrhagic Fever with Renal Syndrome in Republic of Korea” by Park et al. (PNTD-D-20-02190) wherein the authors tested small mammals on Jeju Island during 2018–2020 for orthohantaviruses and provide support for HTNV-specific host use and evolution. The authors provide a compelling argument to support the claim that A. chejuensis -borne HTNV may be a potential etiological agent of HFRS in southern ROK (Republic of Korea) and that the study would bring awareness among physicians for HFRS outbreaks in southern ROK. Additionally, they give attention to the importance of host divergence and geographic isolation events across genetic and geologic evolutionary time.

First concerns:

Though not necessarily a “major” concern, this reviewer feels it is important to mention that taxonomically, Apodemus chejuensis is most likely the sub-species of Apodemus agrarius chejuensis (see https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=754351 and https://www.iucnredlist.org/species/1888/115057408). However, if A. chejuensis is genetically distinct from mainland A. agrarius, the authors could also claim as a new species? (see lines 97-98 S1 Fig).

There may be some push-back from the evolutionary mammalogist community if this is not mentioned. This can be solved in the introduction of A. chejuensis (line 86). Example:

ln 86 “. . . orhtohantavirus, harbored by the subspecies A. agrarius chejuensis (hereafter, A. chejuensis) captured on Jeju Island.”

Check throughout to remain consistent with “orthohantavirus” replacing “hantavirus”. (e.g. lns 66, 69,79,80,86, etc.; it’s hard for this reviewer to get used to also!)

Minor concerns:

Title ln2: might consider adding full subspecies epithet.

ln27: “orthohantaviruses”

ln32: same type of introduction of subspecies (if decided to go that route).; not sure “respectively is needed?

ln35: show (n=x/y) for percentages?

ln55: “significant” might consider different word; could imply the need for statistical evidence.

ln64: taxonomic orders do not need italics; change “Soricomorpha” to “Eulipotyphla”

ln81: change “In” to “On”?

ln94-95: suggested re-write: “The trapped small mammals consisted of 91.4% (64/70) A. chejuensis and 2.8% (2/70) T. triton rodents and 2 C. shantugensis shrews.”

ln102-104: Fig 1 and caption, it might be helpful to place a site number in the text and on the figure.

ln106: consider “Trapping results of small mammals collected at sites on Jeju Island in the Republic of Korea, 2018-2020.”

ln107: bottom of table, “no collection” descriptor is likely not necessary

lns114-115: sentence is a little confusing. It suggests to the reader that the tests (antibodies vs RNA) were exclusive to host and not used for each.

ln135 & 640-641: S1 Table might consider reducing the large number values to 3-4 digits with the conversion metric in column header of the table (e.g. total reads, reads mapped, and depth of coverage).

ln152-153: not sure the last sentence is needed.

ln156: after “positions 644-648” does this refer to S3 Table? May want to include table reference.

ln257: consider a new paragraph starting with the last “The . . .”

ln257-266: The authors make a very compelling argument, and this reviewer agrees with their conclusions.

ln286-289: Again, the authors have provided important information for the medical community as stated in their claims.

ln313: sentence is a little confusing; suggest changing to: “The seroprevalence of orthohantaviruses in rodents demonstrated a higher prevalence of infection in males than females.”

--------------------

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Reviewer #2: No

Reviewer #3: Yes: Matthew T Milholland

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Attachment

Submitted filename: PNTD_HFRS_REVIEW.docx

Click here for additional data file.^{(13.8KB, docx)}

PLoS Negl Trop Dis. 2021 May 12;15(5):e0009400. doi: 10.1371/journal.pntd.0009400.r002

Author response to Decision Letter 0

8 Apr 2021

Attachment

Submitted filename: 4.PNTD-D-20-02190_Minor Revision_20210409.docx

Click here for additional data file.^{(7MB, docx)}

PLoS Negl Trop Dis. doi: 10.1371/journal.pntd.0009400.r003

Decision Letter 1

Brett M Forshey, Emma Wise

22 Apr 2021

Dear Dr. Song,

We are pleased to inform you that your manuscript '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' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Brett M. Forshey

Associate Editor

PLOS Neglected Tropical Diseases

Emma Wise

Deputy Editor

PLOS Neglected Tropical Diseases

***********************************************************

PLoS Negl Trop Dis. doi: 10.1371/journal.pntd.0009400.r004

Acceptance letter

Brett M Forshey, Emma Wise

7 May 2021

Dear Dr. Song,

We are delighted to inform you that your manuscript, "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," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

We have now passed your article onto the PLOS Production Department who will complete the rest of the publication process. All authors will receive a confirmation email upon publication.

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Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

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co-Editor-in-Chief

PLOS Neglected Tropical Diseases

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. A phylogenetic tree based on the mitochondrial DNA cytochrome b gene of Apodemus spp.

(TIF)

Click here for additional data file.^{(5.3MB, tif)}

S2 Fig. Detection of immunoglobulin G (IgG) antibodies against Hantaan virus (HTNV) from Apodemus chejuensis sera and heart fluids by indirect immunofluorescence assay (IFA).

(TIF)

Click here for additional data file.^{(4.2MB, tif)}

S3 Fig. Prediction of N-glycosylation sites in full-length glycoproteins of HTNV and other representative hantaviruses.

(TIF)

Click here for additional data file.^{(4.1MB, tif)}

S1 Table. Summary of mapped reads and depth of coverages by Hantaan virus (HTNV) multiplex PCR-based next-generation sequencing.

(PDF)

Click here for additional data file.^{(107.1KB, pdf)}

S2 Table. Percentage similarity based on L segment of HTNV nucleotide and amino acid sequences between HTNV from Jeju Island and representative rodent-borne orthohantaviruses.

(PDF)

Click here for additional data file.^{(100.7KB, pdf)}

S3 Table. Percentage similarity based on M segment of HTNV nucleotide and amino acid sequences between HTNV from Jeju Island and representative rodent-borne orthohantaviruses.

(PDF)

Click here for additional data file.^{(111.8KB, pdf)}

S4 Table. Percentage similarity based on S segment of HTNV nucleotide and amino acid sequences between HTNV from Jeju Island and representative rodent-borne orthohantaviruses.

(PDF)

Click here for additional data file.^{(111.4KB, pdf)}

S5 Table. Topography and GPS coordinates of the trapping sites.

(PDF)

Click here for additional data file.^{(70.2KB, pdf)}

S6 Table. Oligonucleotide primer sequences generated in this study.

(PDF)

Click here for additional data file.^{(120.4KB, pdf)}

Attachment

Submitted filename: PNTD_HFRS_REVIEW.docx

Click here for additional data file.^{(13.8KB, docx)}

Attachment

Submitted filename: 4.PNTD-D-20-02190_Minor Revision_20210409.docx

Click here for additional data file.^{(7MB, docx)}

Data Availability Statement

The mitochondrial DNA sequences were deposited in GenBank (accession numbers: MW219774- MW2197749). The genomic sequences of HTNV were deposited in GenBank (accession numbers: MW219756- MW219773).

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PERMALINK

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

Kyungmin Park

Won-Keun Kim

Seung-Ho Lee

Jongwoo Kim

Jingyeong Lee

Seungchan Cho

Geum-Young Lee

Jin Sun No

Keun Hwa Lee

Jin-Won Song

Roles

Abstract

Background

Methodology/Principal findings

Conclusions/Significance

Author summary

Introduction

Results

Trapping for small mammals on Jeju Island, ROK

Fig 1. Geography of trapping sites of Hantaan virus (HTNV) collected on Jeju Island, the Republic of Korea.

Table 1. Trapping results of small mammals collected at sites on Jeju Island in the Republic of Korea, 2018–2020.

Serological and molecular prevalence for HTNV collected on Jeju Island

Table 2. Serological and molecular prevalence of Hantaan virus (HTNV) in Apodemus chejuensis captured on Jeju Island from 2018 to 2020.

HTNV RNA loads in tissues from A. chejuensis

Fig 2. Measurement of HTNV RNA loads in different tissues of Apodemus chejuensis, Jeju Island during 2018–2020.

Whole-genome sequencing of HTNV

Table 3. Next-generation sequencing coverages of Hantaan virus (HTNV) from rodents collected in Jeju Island, 2018–2020.

Genomic characterization of HTNV

Phylogenetic and co-evolutionary analyses of HTNV

Fig 3. Phylogeographic analysis of the L, M, and S segments of Hantaan virus (HTNV) collected from rodents in Jeju Island, Republic of Korea (ROK).

Fig 4. Phylogeographic analysis of the L, M, and S segments of Hantaan virus (HTNV) collected from rodents and patients with hemorrhagic fever with renal syndrome (HFRS) in Republic of Korea (ROK).

Fig 5. Tanglegram comparing the phylogenies between hantaviruses and their reservoir hosts in Rodentia.

Genetic reassortment analysis of HTNV

Discussion

Methods

Ethics statement

Sample collection

Mitochondrial DNA (mtDNA) analysis

Indirect immunofluorescence antibody (IFA) test

Reverse transcription-polymerase chain reaction (RT-PCR)

Quantitative PCR (qPCR)

Multiplex PCR-based next-generation sequencing (NGS)

Rapid amplification of cDNA ends (RACE) PCR

Phylogenetic analysis

Co-divergence analysis

Genetic reassortment analysis

N-glycosylation site analysis

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Brett M Forshey

Emma Wise

Roles

Author response to Decision Letter 0

Decision Letter 1

Brett M Forshey

Emma Wise

Roles

Acceptance letter

Brett M Forshey

Emma Wise

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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