Active Targeted Surveillance to Identify Sites of Emergence of Hantavirus
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Clinical Infectious Diseases
MAJOR ARTICLE
Active Targeted Surveillance to Identify Sites of Emergence
of Hantavirus
Won-Keun Kim,1,a Jin Sun No,1 Daesang Lee,2 Jaehun Jung,3 Hayne Park,3 Yongjin Yi,3 Jeong-Ah Kim,1 Seung-Ho Lee,1 Yujin Kim,3 Sunhye Park,2
Seungchan Cho,1 Geum-Young Lee,1 Dong Hyun Song,2 Se Hun Gu,2 Kkothanahreum Park,1 Heung-Chul Kim,4 Michael R. Wiley,5 Patrick S. G. Chain,6
Seong Tae Jeong,2 Terry A. Klein,4 Gustavo Palacios,5 and Jin-Won Song1
1
Department of Microbiology, College of Medicine, Korea University, Seoul, 24th Research and Development Institute, Agency for Defense Development, Daejeon, and 3Armed Forces Medical
Center, Seongnam; 465th Medical Brigade/MEDDAC-Korea, Unit 15281, Seoul; 5Center for Genome Sciences, US Army Medical Research Institute of Infectious Diseases, Maryland; and
6
Bioscience Division, Los Alamos National Laboratory, New Mexico
Background. Endemic outbreaks of hantaviruses pose a critical public health threat worldwide. Hantaan orthohantavirus
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(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 hospitaliza-
tion. 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 geo-
graphic 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 sur-
veillance 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 as-
sessment, and implement preparedness against vector-borne viruses.
Keywords. hantavirus; hemorrhagic fever with renal syndrome; next-generation sequencing; epidemiology; active targeted
surveillance.
Hantaviruses are the causative agent of hemorrhagic fever with Hantaviruses are enveloped, negative-sense, single-stranded,
renal syndrome (HFRS) and hantavirus pulmonary syndrome RNA viruses that harbor large (L), medium (M), and small
(HPS) in humans [1]. In East Asia, approximately 150 000 (S) genome segments [4] and are considered part of the genus
HFRS cases due to Hantaan orthohantavirus (HTNV) and Orthohantavirus of the family Hantaviridae of the order
Seoul orthohantavirus infections are reported annually, with Bunyavirales. The L segment encodes for an RNA-dependent
fatality rates of 1%–15% [2]. In the Republic of Korea (ROK), RNA polymerase, while the M segment contains membrane
300–600 HFRS cases are reported annually, with a mean case glycoproteins (Gn and Gc). The S segment encodes for a nu-
fatality rate of 1%–4% [3]. Despite the high burden of disease cleocapsid protein. In 1978, HTNV was first identified in
due to hantaviruses, no effective vaccines or specific therapies the ROK as the etiologic agent of HFRS [5]. The striped field
are readily available. mouse (Apodemus agrarius) has been proposed as the natural
reservoir for HTNV. The transmission of HTNV from rodents
Received 5 September 2018; editorial decision 11 March 2019; accepted 19 March 2019; to humans typically occurs through the inhalation of infected
published online March 20, 2019.
a
Current address: Department of Microbiology, College of Medicine, Hallym University, aerosolized particles. Although some nosocomial infections
Chuncheon have been described in South American HPS cases, human-to-
Correspondence: J.-W. Song, Korea University, College of Medicine, Department of human transmission events have been rarely reported [6, 7]. The
Microbiology, 73 Inchon-ro, Seongbuk-gu, Seoul 02841, Republic of Korea (jwsong@korea.
ac.kr). incubation period of HTNV infections is known to be up to 2–3
Clinical Infectious Diseases® 2020;70(3):464–73 weeks in humans [8]. The onset of HFRS was reported to be as
© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society
of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.
long as 41 days. The typical HFRS clinical course includes the
DOI: 10.1093/cid/ciz234 following 5 phases: febrile, hypotensive, oliguric, diuretic, and
464 • cid 2020:70 (1 February) • Kim et al
convalescent. The febrile phase is an early stage that involves (AFCH18-IRB-004) with approvals for all aspects of human
fever, pains, and edema for 3–5 days. The hypotensive phase participants and case studies. Live trapping of rodents at mil-
lasts for a few hours and is characterized by internal bleeding, itary training sites and HFRS-endemic areas was conducted in
reduced blood pressure, thrombocytopenia, and proteinuria. accordance with United States Forces Korea Regulation 40–1,
The oliguric phase (decreased urine output) lasts for 3–7 days Prevention, Surveillance, and Treatment of Hemorrhagic Fever
and is defined by renal dysfunction, blood electrolyte imbal- with Renal Syndrome. Rodents were transported to Korea
ance, and hypervolemia. The diuretic (increased urine output) University where they were under isoflurane anesthesia and
and convalescent phases are recovery stages that last for several euthanized by cardiac puncture; tissues were collected in ac-
weeks to months, marked by urine output control, progressive cordance with procedures approved by KU-IACUC (2010–212
improvements in glomerular filtration rate, and renal blood flow. and 2016–0049) protocol.
Several studies have investigated the genomic and
phylogeographic relationships between HFRS/HPS patients HFRS Case Description
and mammalian reservoirs of endemic hantaviruses [9–12]. On 12 December 2016, patient ROK Army (ROKA) 16-9 was
identified after a separate military training facility in Dosin-ri,
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However, the majority of these studies only characterized
a fragment of the hantavirus genome, which could limit Yeoncheon-gun, Gyeonggi Province (Figure 1 and Table 1).
phylogeographical and clinical analyses due to genetic diver- An outbreak of HFRS was detected in Gyeonggi Province in
sity and exchanges, that is, detecting a genetic variant, recom- January 2017, involving 4 military patients from the ROK Army:
bination, or reassortment [13–15]. Recently, complete genomes ROKA17-3 on 8 January, ROKA17-5 on 12 January, ROKA17-7
of HTNV from the ROK were sequenced using single-primer on 13 January, and ROKA17-8 on 14 January. ROKA17-3 was
amplification and also a targeted enrichment approach using assigned to Yangpyeong-gun, while ROKA17-5, ROKA17-7,
next-generation sequencing (NGS) [16, 17]. However, neither and ROKA17-8 were assigned to Yangju-si. Inspection of their
of these studies actively investigated clinical cases of HFRS clinical history revealed that the 4 soldiers had conducted a
and their spatial–temporal relationship to zoonotic strains of joint military exercise in Paju-si, Gyeonggi Province, on 17–21
HTNV. Here, we integrated clinical and epidemiological data December 2016. After the training, the patients returned to
from 5 patients with HFRS and subsequently several HTNV- their assigned military bases. On 8–14 January, ROKA17-3,
positive rodents to identify potential sites of zoonotic transmis- ROKA17-5, ROKA17-7, and ROKA17-8 sequentially presented
sion of HTNV in the northern provinces of the ROK. the onset of HFRS clinical symptoms and were hospitalized. On
the basis of epidemiological interviews, ROKA16-9, ROKA17-
3, ROKA17-7, and ROKA17-8 were not immunized. ROKA17-5
METHODS
was vaccinated twice, although he was not satisfied with the
Ethics Statement hantavirus immunization program [18].
This study was performed in accordance with the ethical
guidelines for the Korea University Institutional Animal Patient Specimen Collection
Care and Use Committee (KU-IACUC), Korea University. Five HFRS patients were selected based on the following criteria:
Each human sample was collected under informed consent patient was infected with HTNV, an epidemiological interview
Table 1. Summary of Hemorrhagic Fever With Renal Syndrome Patient Sample Information
Date Sample Date of Labora- Training
Patient Onset Collected tory Diagnosisa Date of Outdoor Activity Activity Location
ROKA16-9 12 December 14 December 16 December 2016 30 November 2016–2 December 2016 Military training Dosin-ri
2016 2016 6 December 2016–7 December 2016 (stakeout in Yeoncheon-gun
the hills)
ROKA17-3 8 January 2017 13 January 2017 16 January 2017 17 December 2016–21 December 2016 Military training Mugeon-ri
(stakeout in Paju-si
the hills)
ROKA17-5 12 January 2017 14 January 2017 16 January 2017 17 December 2016–21 December 2016 Military training Mugeon-ri
(stakeout in Paju-si
the hills)
ROKA17-7 13 January 2017 17 January 2017 23 January 2017 17 December 2016–21 December 2016 Military training Mugeon-ri
(stakeout in Paju-si
the hills)
ROKA17-8 14 January 2017 18 January 2017 23 January 2017 17 December 2016–21 December 2016 Military training Ohyeon-ri
(stakeout in Paju-si
the hills)
a
Laboratory diagnosis was done using indirect immunofluorescence antibody test and reverse transcription-polymerase chain reaction as shown in Table 3.
Active Surveillance of Hantavirus • cid 2020:70 (1 February) • 465
was included, and active rodent surveillance was conducted. infection, and Hantavax vaccination [23]. The medical charts
Whole blood or serum samples from HFRS patients were pro- were reviewed.
vided by the Korea Armed Forces Capital Hospital, Gyeonggi
Province, ROK. Serum was extracted from whole blood from Targeted Rodent Trapping
HFRS patients by centrifuging at 4000 rpm at 4°C for 5 minutes Rodents (A. agrarius, Myodes regulus, and Mus musculus)
and then stored at –80°C. and soricomorphs (shrews; Crocidura lasiura) were captured
at Ohyeon-ri (ri = village) and Mugeon-ri, Paju-si (si = city
Clinical Classification of HFRS Patients area), and Dosin-ri, Yeoncheon-gun (gun = region), Gyeonggi
HFRS patients were classified as “mild,” “moderate,” and “se- Province in ROK. Sherman collapsible live traps (8 × 9 × 23 cm;
vere” [19, 20]. Mild was marked by kidney injury without oli- H. B. Sherman, Tallahassee, FL) were placed at 4- to 5-m
guria and hypotension. Moderate was characterized by uremia, intervals and collected over a 1- to 3-day period. For each day,
pulmonary edema, hypotension, hemorrhage (petechiae and
100 traps were set up. In Figure 1, trapping locations are shown
conjunctival injection), acute kidney injury (AKI) with typical
and marked by various mouse patterns.
oliguria, white blood cell count ≥14 000/μL, and days of fever
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>38°C. Moderate was characterized by hypotension ≤95 mmHg, Indirect Immunofluorescence Antibody Test
hypertension ≥141 mmHg, maximum urine volume ≥3500 mL/ Serum, diluted 1:32, was added to wells containing HTNV-
day, minimum platelets ≤89 000/μL, and serum albumin ≤3.0/
infected Vero E6 cells. The cells were incubated at 37°C for
dL [21, 22]. Severe was defined as having moderate indications
30 minutes and then washed with phosphate-buffered saline.
along with 1 or more clinical complications including refractory
A total of 25 µL fluorescein isothiocyanate–conjugated goat
shock, visceral hemorrhage, heart failure, transfusion, hemodi-
antihuman and mouse immunoglobulin G (IgG) antibodies
alysis, and AKI with oliguria (urine output of 50–500 mL/day for
(ICN Pharmaceuticals, Laval, Canada) were added to each
≤5 days) or anuria (urine output of 38°C Yes Yes Yes Yes Yes
White blood cell count (≥14 000/μL) No Yes Yes No Yes
Hypotensive Minimum systolic BP (≤95 mmHg) Yes Yes Yes Yes Yes
Serum albumin (≤3.0/dL) No No Yes Yes No
Minimum platelets (≤89 000/μL) No Yes Yes Yes Yes
Oliguric and diuretic Maximum systolic BP (≥141 mmHg) Yes Yes Yes Yes Yes
Maximum urine volume (≥3500 mL/day) Yes Yes Yes No Yes
Minimum urine volume (≤500 mL/day) No No Yes No No
Estimated glomerular filtration rate (≤15 mL/min) No Yes Yes No Yes
Event Transfusion No No No Yes No
Hemodialysis No No No No No
Total 4/11 7/11 9/11 6/11 7/11
Abbreviation: BP, blood pressure.
466 • cid 2020:70 (1 February) • Kim et al
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Figure 1. Geographic map of Hantaan orthohantavirus (HTNV) strains from hemorrhagic fever with renal syndrome (HFRS) patients and rodents. Sites of HFRS outbreaks
and HTNV-specific sero- and reverse transcription-polymerase chain reaction–positive Apodemus agrarius collections are shown. HTNV-infected rodents are marked with
various patterns. Patient ROKA16-9 was assigned to and trained at Dosin-ri, Yeoncheon-gun. A star (*) indicates Mugeon-ri and Ohyeon-ri, Paju-si. Patients ROKA17-3,
ROKA17-5, ROKA17-7, and ROKA17-8 conducted joint military training on 21–26 December 2016. Red arrows indicate joint military training on 21 December. Blue arrows indi-
cate return from training sites to the assigned military base on 26 December 2016. Paju-si, Yeoncheon-gun, Yangju-si, Namyangju-si, and Pocheon-si are located in Gyeonggi
Province; Cheorwon-gun, Hwacheon-gun, and Yanggu-gun are located in Gangwon Province.
Real-time PCR was determined for HTNV L (1-6530 nt), M (1-3616 nt), and
RNA loads were examined by targeting the HTNV S segment S segments (1-1696 nt), which were TN93+G, T92+G, and
with SYBR Green PCR Master Mix (Applied Biosystems) in a T92+G, respectively. Maximum likelihood method was used
Quantstudio 6 Flex Real-time PCR System (Applied Biosystems) to generate the phylogenetic trees using MEGA 6.0. Topologies
[25]. PCR was conducted by a cycle of 95°C for 10 minutes, were assessed by bootstrap analysis of 1000 iterations. For
followed by 45 cycles of 95°C for 15 seconds and 60°C for 1 minute. phylogeographic analyses, the tracking of HTNV genomes from
HFRS patients was examined by epidemiological investigations,
Multiplex PCR-based NGS phylogenies of the viruses, and geographic locations of HTNV-
cDNA was enriched by using HTNV-specific primer mixtures infected rodents.
and Solg 2× Uh-Taq PCR Smart mix (Solgent, Seoul, ROK) as
previously described [17]. The libraries were prepared using a
RESULTS
TrueSeq Nano DNA LT sample preparation kit (Illumina, San
Diego, CA) according to the manufacturer’s instructions. NGS Laboratory Diagnosis and Multiplex PCR-based NGS
was performed on a MiSeq benchtop sequencer (Illumina) Serum from ROKA16-9 patient was collected on 14 December
with 2 × 150 bp using a MiSeq reagent V2 (Illumina). Illumina 2016, and laboratory diagnosis confirmed HTNV infection by
FASTQ files were extracted and analyzed using Empowering indirect immunofluorescence antibody test (IFA) and RT-PCR
the Development of Genomics Expertise [26]. on 16 December 2016 (Table 3). IFA and RT-PCR tests confirmed
the presence of anti-HTNV IgG and HTNV RNA of sera from
Phylogenetic Analysis patients ROKA17-3, ROKA17-5, ROKA17-7, and ROKA17-8
Multiple sequences of HTNV were aligned using the MUSCLE on 16–23 January 2017. Quantitation of HTNV RNA in HFRS
algorithm in MEGA 6.0 [27]. The best-fit substitution model patient sera was determined by real-time PCR. The cycle
Active Surveillance of Hantavirus • cid 2020:70 (1 February) • 467Table 3. Summary of Laboratory Diagnosis and Multiplex Polymerase Chain Reaction–based Next-generation Sequencing for Hemorrhagic Fever With
Renal Syndrome Patients
Coverage of HTNV Genomic Sequences
by Multiplex PCR-based Next-generation
Sequencingb
Patient IFA for Anti-HTNV Immunoglobulin Ga RT-PCR for HTNV RNA Ct Value L Segment M Segment S Segment
ROKA16-9 1:512 + 24.1 99.8 99.2 99.2
ROKA17-3 1:64 + 30.2 98.6 99.2 99.1
ROKA17-5 1:32 + 29.7 99.9 98.1 99.1
ROKA17-7 …c + 29.3 99.9 98.9 99.0
ROKA17-8 1:32 + 35.2 98.6 98.7 98.9
The plus sign (+) indicates positivity.
Abbreviations: Ct, cycle threshold; HTNV, Hantaan orthohantavirus; IFA, indirect immunofluorescence antibody test; RT-PCR, reverse transcription-polymerase chain reaction.
a
Antibody titration was tested by serial 2-fold dilution from 1:32 diluted serum.
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b
Genome coverages were determined by obtained consensus sequence matching to genome positions of HTNV 76–118 strain (GenBank accession numbers: L segment, NC_005222; M
segment, NC_005219; S segment, NC_005218).
c
The titer of anti-HTNV immunoglobulin G was undetectable.
threshold (Ct) values of HTNV RNA for patients ROKA16-9, M segment did not show a clear match, and the S segment
ROKA17-3, ROKA17-5, ROKA17-7, and ROKA17-8 were 24.1, sequences formed a genetic group with HTNV strains previ-
30.2, 29.7, 29.3, and 35.2, respectively. Patient ROKA16-9 had ously detected at Jangjwa-ri, Paju-si (Supplementary Figures
the lowest Ct value (24.1), indicating the highest titer among 1–3). HTNV from HFRS patients ROKA17-3, ROKA17-5,
the patients surveyed. ROKA17-7, and ROKA17-8 formed a genetic cluster with
Whole-genome sequences (WGSs) of HTNV from patient HTNV identified in Ohyeon-ri, Paju-si. However, based on
sera were recovered by performing multiplex PCR-based NGS. retrospective epidemiological surveys, an incongruence was
The coverage rates of HTNV tripartite RNA genomic sequences observed; patients ROKA17-3, ROKA17-5, and ROKA17-7
ranged from 98.6% to 99.9% in the HFRS patients. The genomic executed joint military training at Mugeon-ri, whereas patient
sequences of 3’ and 5’ ends were empirically determined due to ROKA17-8 conducted training at Ohyeon-ri.
the conserved region of the family Hantaviridae. The HTNV To solve these discrepancies, targeted rodent trapping was
sequences were deposited in GenBank (MH598466-MH598507). conducted on 28–29 March 2017 at Mugeon-ri, Paju-si where
patients ROKA17-3, ROKA17-5, and ROKA17-7 conducted
Active Targeted Rodent Surveillance military training. A total of 30 small mammals were captured,
The phylogenetic analyses of the viral genome recovered from including 29 (96.7%) A. agrarius and 1 (3.3%) C. lasiura
patient ROKA16-9 demonstrated that the L segment formed (Table 4). Six (20.7%) of 29 A. agrarius sera were positive for
a genetic cluster with the HTNV strain from Yanggu-gun, the anti-HTNV IgG. Three (50.0%) of the 6 lung tissues of the
Table 4. Summary of Targeted Rodent Trapping and Laboratory Screening of Hantaan Orthohantavirus
RT-PCR for HTNVb
Number Seropositivity for Anti-HTNV Seropositive Seronegative
Date Trapping Site Species Captured Immunoglobulin G by IFAa Animals Animals
28–29 March 2017 Mugeon-ri, Paju-si Apodemus agrarius 29 6/29 (20.7%) 3/6 (50.0%) 0/23
Crocidura lasiura 1 0/1 … 0/1
Ohyeon-ri, Paju-si A. agrarius 12 1/12 (8.3%) 0/1 0/11
Subtotal 42 7/42 (16.7%) 3/7 (42.8%) 0/35
18–20 April 2017 Dosin-ri, Yeoncheon- A. agrarius 17 4/17 (23.5%) 4/4 (100%) 0/13
gun
C. lasiura 3 0/3 … 0/3
Myodes regulus 5 0/5 … 0/5
Mus musculus 2 0/2 … 0/2
Subtotal 27 4/27 (14.8%) 4/4 (100%) 0/23
Abbreviations: HTNV, Hantaan orthohantavirus; IFA, indirect immunofluorescence antibody test; RT-PCR, reverse transcription-polymerase chain reaction.
a
Indirect immunofluorescence antibody test for anti-HTNV immunoglobulin G antibody.
b
RT-polymerase chain reaction for M segment (1970–2342 nt) using HTNV-specific primers.
468 • cid 2020:70 (1 February) • Kim et alseropositive rodents were positive by RT-PCR for HTNV DISCUSSION
RNA. Anti-HTNV IgG was found in 1 (8.3%) of 12 A. agrarius The recent outbreak and spread of Ebola, Zika, and Middle East
captured in Ohyeon-ri, Paju-si. However, none of the rodents respiratory syndrome viruses have increased enhanced caution
carried HTNV RNA. and boosted the need for active surveillance, diagnostics, and
Additional targeted small mammal trapping was conducted real-time tracing of emerging infectious agents using high-
on 18–20 April 2017 at Dosin-ri, Yeoncheon-gun. A total of 27
throughput technologies [28, 29]. In this study, we combined
rodents and soricomorphs were captured, including 17 (63.0%)
clinical, epidemiological, and NGS data to investigate and track
A. agrarius, 3 (11.1%) C. lasiura, 5 (18.5%) M. regulus, and 2
the infectious source of 5 HFRS patients in 2016–2017; all of the
(7.4%) M. musculus. Anti-HTNV IgG was detected in sera of
patients participated in off-site military exercises within 30 days
4/17 (23.5%) A. agrarius, whereas none of the other rodents and
prior to the onset of clinical symptoms. These patients resided
shrews were seropositive. All 4 lung tissues of the seropositive
in the northern provinces of the ROK. These areas have been the
A. agrarius were positive for HTNV RNA by RT-PCR.
focal point of several previous studies of hantaviruses. In 2009,
4 US soldiers were diagnosed with HFRS by HTNV through
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High-resolution Phylogeographic Analyses of HTNV From HFRS Patients
and Rodents partial genome sequencing and phylogeographical inferences
WGSs of HTNV were recovered from the lung tissues of [10]. Furthermore, multiplex PCR-based NGS enabled nearly
A. agrarius (Aa17-7 and Aa17-8) captured at Mugeon-ri, complete HTNV genome sequencing from 2 ROK soldiers and
Paju-si, using multiplex PCR-based NGS (Table 5). The ge- 2 US Army soldiers from 2013 to 2015 [17].
nome sequences of HTNV Aa17-48, Aa17-49, and Aa17-53 We applied a high-resolution NGS approach to associate
were almost completely obtained from seropositive A. agrarius 5 HFRS cases to specific training sites based on published
captured at Dosin-ri, Yeoncheon-gun. The 3’ and 5’ termini ge- sequencing data from the same geographical regions. Our in-
nomic sequences were empirically replaced with the conserved itial phylogenetic inference revealed incongruences between
sequence of the family Hantaviridae. the patients’ physical locations and the clustering of obtained
The phylogeographic analysis showed that HTNV obtained HTNV strains. Patient ROKA16-9 sequence clustered with ro-
from patient ROKA16-9 showed that the L, M, and S segments dent sequences from different sites, but rodents had never been
formed a homologous genetic group with HTNV newly ac- captured at the Dosin-ri, Yeoncheon-gun. The phylogeny of the
quired from A. agrarius (Aa17-48, Aa17-49, and Aa17-53) HTNV sequences from ROKA17-3, ROKA17-5, and ROKA17-7
at Dosin-ri, Yeoncheon-gun (Figures 2–4). HTNV obtained genetically clustered with each other, while ROKA17-8 formed
from patients ROKA17-3, ROKA17-5, and ROKA17-7 formed a clustering with rodents captured at Ohyeon-ri, Paju-si.
a genetic lineage with HTNV from A. agrarius captured at Epidemiological interviews with patients ROKA17-3, ROKA17-
Mugeon-ri, Paju-si, where the ROK soldiers had trained. The 5, and ROKA17-7 demonstrated that they resided at Mugeon-ri
L and M segments of HTNV observed in patient ROKA17-8 in Paju-si during the training period; rodents had never been
grouped with HTNV from Ohyeon-ri in Paju-si, whereas the S collected from this region. However, ROKA17-8 conducted the
segment aligned with HTNV strains from Mugeon-ri. military training at Ohyeon-ri in Paju-si.
Table 5. Summary of Laboratory Diagnosis and Multiplex Polymerase Chain Reaction–based Next-generation Sequencing for Hantaan Orthohantavirus
From Rodents Captured at Military Training Sites Where Republic of Korea Soldiers Most Likely Acquired Hemorrhagic Fever With Renal Syndrome
Coverage of HTNV Genomic Sequences
by Multiplex PCR-based Next-generation
Sequencinga
Rodent Sample Location IFA for Anti-HTNV IgG RT-PCR for HTNV RNA Ct Value S Segment M Segment L Segment
Aa17-6 Mugeon-ri, Paju-si 1:1024 + 35.7 93.0 51.9 56.3
Aa17-7 Mugeon-ri, Paju-si 1:2048 + 32.0 99.0 99.7 99.7
Aa17-8 Mugeon-ri, Paju-si 1:512 + 21.5 99.0 99.7 99.7
Aa17-48 Dosin-ri, Yeoncheon-gun 1:512 + 24.7 99.5 99.4 99.8
Aa17-49 Dosin-ri, Yeoncheon-gun 1:128 + 21.5 99.0 99.3 99.7
Aa17-52 Dosin-ri, Yeoncheon-gun 1:1024 + 27.9 99.0 97.7 99.7
Aa17-53 Dosin-ri, Yeoncheon-gun 1:512 + 26.5 99.1 99.2 99.7
The plus sign (+) indicates positivity.
Abbreviations: Ct, cycle threshold; IFA, indirect immunofluorescence antibody test; IgG, immunoglobulin G; HTNV, Hantaan orthohantavirus; RT-PCR, reverse transcription-polymerase chain
reaction.
a
Genome coverages were determined by obtained consensus sequence matching to genome positions of HTNV 76–118 strain (GenBank accession numbers: L segment, NC_005222;
M segment, NC_005219; S segment, NC_005218).
Active Surveillance of Hantavirus • cid 2020:70 (1 February) • 469Downloaded from https://academic.oup.com/cid/article/70/3/464/5397012 by guest on 30 December 2020 Figure 2. High-resolution phylogeographic analysis of Hantaan orthohantavirus (HTNV) L segment from hemorrhagic fever with renal syndrome (HFRS) outbreaks after active targeted surveillance. Whole-genome sequences of HTNV from HFRS patients and lung tissues of seropositive Apodemus agrarius were obtained by multiplex poly- merase chain reaction–based next-generation sequencing. The phylogenetic tree of HTNV L segments (1–6530 nt) is shown using the TN93 (Tamura-Nei) model+G model. Bold red indicates HTNV strains identified from HFRS patients in Paju-si. Bold blue indicates an HTNV strain identified from HFRS patient, Yeoncheon-gun. Red and blue squares indicate high-resolution phylogeographic analysis of HFRS outbreaks in Paju-si and Yeoncheon-gun, respectively. Paju-si, Yeoncheon-gun, Dongducheon-si, and Pocheon-si are located in Gyeonggi Province; Yanggu-gun is located in Gangwon Province. To clarify the infectious source for patients ROKA16-9, ROKA17-5, and ROKA17-7 who trained at Mugeon-ri formed ROKA17-3, ROKA17-5, and ROKA17-7, we applied active ro- a genetic lineage with a zoonotic HTNV from A. agrarius dent surveillance and capturing in areas where each of these captured from that same site. patients resided prior to their illness, specifically at Dosin-ri Previous phylogeographic analyses have demonstrated in Yeoncheon-gun and Mugeon-ri in Paju-si training sites. The that it is possible to link viruses from HFRS patients to their HTNV sequences acquired from collected rodents allowed us to reservoir hosts in order to indicate the most likely site of in- infer a posteriori the reported sites of HTNV infection for the 5 fection [17]. Our findings strengthen this case, as zoonotic symptomatic patients. For example, the targeted small mammal events can be retrospectively investigated to clarify epidemi- trapping at Yeoncheon-gun consistently demonstrated that ological findings at a higher resolution using active surveil- the ROKA16-9 HTNV sequence clustered with rodent HTNV lance. To prevent HFRS from becoming a public health threat, from the military training site at Dosin-ri. Furthermore, we HFRS-endemic locations were cleaned and organized to avoid demonstrated that the HTNV from HFRS patients ROKA17-3, contacts with rodents. Public warning signs were installed near 470 • cid 2020:70 (1 February) • Kim et al
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Figure 3. High-resolution phylogeographic analysis of Hantaan orthohantavirus (HTNV) M segment from hemorrhagic fever with renal syndrome (HFRS) outbreaks after
active targeted surveillance. Whole-genome sequences of HTNV from HFRS patients and lung tissues of seropositive Apodemus agrarius were obtained by multiplex
polymerase chain reaction–based next-generation sequencing. The phylogenetic tree of HTNV M segments (1–3616 nt) was generated on the basis of the T92 (Tamura
3-parameter) model+G model. Bold red indicates HTNV strains identified from HFRS patients in Paju-si. Bold blue indicates an HTNV strain identified from HFRS patient,
Yeoncheon-gun. Red and blue squares indicate high-resolution phylogeographic analysis of HFRS outbreaks in Paju-si and Yeoncheon-gun, respectively. Paju-si, Yeoncheon-
gun, Dongducheon-si, and Pocheon-si are located in Gyeonggi Province; Yanggu-gun is located in Gangwon Province.
the endemic areas identified in this study. In addition, military segments grouped to HTNV from Ohyeon-ri, Paju-si, whereas
exercises were rescheduled to avoid the highly endemic season. the S segment clustered with HTNV from Mugeon-ri. The ge-
This study suggests that individuals at high risk of contracting nomic characteristics might be suggestive active exchange of
HFRS should be vaccinated according to the hantavirus im- genetic components, but recombination and reassortment
munization program [23, 30]. These strategies (posteriori and analyses were considered insignificant based on Recombination
priori interventions) may have an impact on the prevention of Detection Program 4 and Graph-incompatibility-based
emerging or reemerging hantavirus outbreaks and the reduc- Reassortment Finder’ (not shown). Dynamic genetic exchanges
tion of HFRS incidence. were observed from HTNV and Imjin virus in the ROK [13, 14,
This study had limitation due to a possible technical issue 17]. Development of bioinformatics approaches may facilitate
or insufficient HTNV genomic sequences. The phylogeny of the detection of genetic exchange events in viruses. Continuous
ROKA16-9 was highly diversified, demonstrating that the L, animal trapping and addition of HTNV genomic sequences
M, and S segments shared common ancestors with different from HFRS-endemic areas may help in clarification of the
HTNV strains. Phylogenetic patterns of ROKA17-8 L and M phylogeography.
Active Surveillance of Hantavirus • cid 2020:70 (1 February) • 471Downloaded from https://academic.oup.com/cid/article/70/3/464/5397012 by guest on 30 December 2020
Figure 4. High-resolution phylogeographic analysis of Hantaan orthohantavirus (HTNV) S segment from hemorrhagic fever with renal syndrome (HFRS) outbreaks after ac-
tive targeted surveillance. Whole-genome sequences of HTNV from HFRS patients and lung tissues of seropositive Apodemus agrarius were obtained by multiplex polymerase
chain reaction–based next-generation sequencing. The phylogenetic tree of HTNV S segments (1–1696 nt) is shown on the basis of the T92 (Tamura 3-parameter) model+G
model. Bold red indicates HTNV strains identified from HFRS patients in Paju-si. Bold blue indicates an HTNV strain identified from HFRS patient, Yeoncheon-gun. Red and
blue squares indicate high-resolution phylogeographic analysis of HFRS outbreaks in Paju-si and Yeoncheon-gun, respectively. Paju-si, Yeoncheon-gun, Dongducheon-si, and
Pocheon-si are located in Gyeonggi Province; Yanggu-gun is located in Gangwon Province.
In conclusion, whole genomic sequencing, epidemiolog- control strategies of hantavirus-borne diseases to prevent
ical surveys, and active targeted surveillance enabled high- endemic transmission.
resolution phylogeographic analysis of HFRS and allowed
for the identification of the putative HTNV infection site. Supplementary Data
This was done by temporally and spatially tracking ge- Supplementary materials are available at Clinical Infectious Diseases
online. Consisting of data provided by the authors to benefit the reader,
nomic sequences of HTNV obtained from HFRS patients the posted materials are not copyedited and are the sole responsibility of
with viral data obtained from reservoir host rodents. This the authors, so questions or comments should be addressed to the corre-
novel approach promises to provide critical insights into sponding author.
continued surveillance of viral infections, which will lead
Notes
to the development of a precise phylogeographic analysis
Acknowledgments. The authors thank Dr Nicholas Di Paola for his con-
and disease risk analyses and preparedness. The epide-
tribution to the editing of the manuscript.
miological association of patients with infectious sources Financial support. This work was supported by the Agency for Defense
will aid in the identification, disease risk assessment, and Development (UD160022ID); the Research Program To Solve Social Issues of
472 • cid 2020:70 (1 February) • Kim et althe National Research Foundation of Korea (NRF) funded by the Ministry of 14. Lee SH, Kim WK, No JS, et al. Dynamic circulation and genetic exchange of a shrew-
Science and Information and Communication Technology (ICT) (NRF- borne hantavirus, Imjin virus, in the Republic of Korea. Sci Rep 2017; 7:44369.
2017M3A9E4061992) and the Armed Forces Health Surveillance 15. Li D, Schmaljohn AL, Anderson K, Schmaljohn CS. Complete nucleotide
sequences of the M and S segments of two hantavirus isolates from California: ev-
Branch, Global Emerging Infections Surveillance and Response System
idence for reassortment in nature among viruses related to hantavirus pulmonary
(P0025-15-ME).
syndrome. Virology 1995; 206:973–83.
Potential conflicts of interest. All authors: No reported conflicts of in- 16. Song DH, Kim WK, Gu SH, et al. Sequence-independent, single-primer ampli-
terest. All authors have submitted the ICMJE Form for Disclosure of fication next-generation sequencing of Hantaan virus cell culture-based isolates.
Potential Conflicts of Interest. Conflicts that the editors consider relevant to Am J Trop Med Hyg 2017; 96:389–94.
the content of the manuscript have been disclosed. 17. Kim WK, Kim JA, Song DH, et al. Phylogeographic analysis of hemorrhagic
fever with renal syndrome patients using multiplex PCR-based next generation
sequencing. Sci Rep 2016; 6:26017.
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