microorganisms

Article
Molecular Characterization of Dengue Virus Strains from the
2019–2020 Epidemic in Hanoi, Vietnam
Juthamas Phadungsombat 1 , Huong Thi Thu Vu 2 , Quynh Thi Nguyen 3 , Ha Thi Van Nguyen 2 , Ha Thi
Nhu Nguyen 2 , Bich Thi Dang 2 , Emi E. Nakayama 1 , Azumi Ishizaki 3 , Hiroshi Ichimura 3 , Tatsuo Shioda 1, *
and Thach Ngoc Pham 2

1 Department of Viral Infections, Research Institute for Microbial Diseases, Osaka University,
Osaka 565-0871, Japan; [email protected] (J.P.); [email protected] (E.E.N.)
2 National Hospital for Tropical Disease, Hanoi 100000, Vietnam; [email protected] (H.T.T.V.);
[email protected] (H.T.V.N.); [email protected] (H.T.N.N.); [email protected] (B.T.D.);
[email protected] (T.N.P.)
3 Department of Viral infection and International Health, Graduate School of Medical Science, Kanazawa
University, Kanazawa 920-8640, Japan; [email protected] (Q.T.N.);
[email protected] (A.I.); [email protected] (H.I.)
* Correspondence: [email protected]

Abstract: Dengue virus (DENV), which has circulated in Vietnam for several decades, has multiple
serotypes and genotypes. A 2019 dengue outbreak resulted in a larger number of cases than any other
outbreak. We conducted a molecular characterization using samples collected in 2019–2020 from
dengue patients in Hanoi and nearby cities located in northern Vietnam. The circulating serotypes
were DENV-1 (25%, n = 22) and DENV-2 (73%, n = 64). Phylogenetic analyses revealed that all
DENV-1 (n = 13) were genotype I and clustered to local strains circulating during the previous
outbreak in the 2017, whereas DENV-2 consisted of two genotypes: Asian-I (n = 5), related to local
Citation: Phadungsombat, J.; Vu, strains from 2006–2022, and cosmopolitan (n = 18), the predominant genotype in this epidemic.
H.T.T.; Nguyen, Q.T.; Nguyen, H.T.V.; The current cosmopolitan virus was identified as having an Asian-Pacific lineage. The virus was
Nguyen, H.T.N.; Dang, B.T.; closely related to strains in other recent outbreaks in Southeast Asian countries and China. Multiple
Nakayama, E.E.; Ishizaki, A.; introductions occurred in 2016–2017, which were possibly from maritime Southeast Asia (Indonesia,
Ichimura, H.; Shioda, T.; et al.
Singapore, and Malaysia), mainland Southeast Asia (Cambodia and Thailand), or China, rather than
Molecular Characterization of
from an expansion of localized Vietnamese cosmopolitan strains that were previously detected in
Dengue Virus Strains from the
the 2000s. We also analyzed the genetic relationship between Vietnam’s cosmopolitan strain and
2019–2020 Epidemic in Hanoi,
recent global strains reported from Asia, Oceania, Africa, and South America. This analysis revealed
Vietnam. Microorganisms 2023, 11,
1267. https://doi.org/10.3390/
that viruses of Asian-Pacific lineage are not restricted to Asia but have spread to Peru and Brazil in
microorganisms11051267 South America.

Academic Editors: Shengxi Chen and

Keywords: DENV-1; DENV-2; DENV genotype; phylogenetic tree; Vietnam; Hanoi
Fabio Zicker

Received: 19 April 2023

Revised: 9 May 2023
Accepted: 10 May 2023 1. Introduction
Published: 11 May 2023 Dengue is a mosquito-borne disease transmitted to humans via the bite of infected
mosquitos and commonly occurs in over 100 tropical and subtropical countries. Clinically,
dengue cases vary in severity from mild to fatal. The World Health Organization (WHO)
categorizes the disease as dengue (with or without warning signs) and severe dengue [1].
Copyright: © 2023 by the authors.
Symptoms, such as high fever, severe headache, muscle and joint pain, nausea, and rash,
Licensee MDPI, Basel, Switzerland.
This article is an open access article
manifest for 2–7 days, and warning signs of severe disease include severe abdominal pain,
distributed under the terms and
persistent vomiting, rapid breathing, bleeding gums, and nosebleed [2]. The incidence of
conditions of the Creative Commons dengue cases reported to the WHO increased from approximately 500,000 cases in 2009 to
Attribution (CC BY) license (https:// 5 million cases in 2019 [2], resulting in significant global human and economic impacts.
creativecommons.org/licenses/by/ The causative agent of dengue fever is dengue virus (DENV) (genus Flavivirus, family
4.0/). Flaviviridae). DENVs can be classified into four serotypes (DENV-1, DENV-2, DENV-3, and

Microorganisms 2023, 11, 1267. https://doi.org/10.3390/microorganisms11051267 https://www.mdpi.com/journal/microorganisms

Microorganisms 2023, 11, 1267 2 of 16

DENV-4). In primary DENV infection, long-term host immunity is specifically generated

against the actual serotype, with short-term protection against other serotypes. Subsequent
secondary infection with a different serotype can lead to antibody-dependent enhancement,
increasing the risk of severe dengue [3]. Within the dengue serotypes, 4–6 genotypes have
been genetically defined [4]. Serotype/genotype replacement has occurred concurrently
with the increase in the number of patients [5].
Vietnam, located in Southeast Asia and bordered by Laos, Cambodia, and China, is a
hyperendemic country for dengue. Periodic and cyclic DENV circulation patterns have
occurred in Vietnam, but DENV-1 and -2 are the most common serotypes [5,6]. In 2019, the
highest recorded number of dengue cases was reported worldwide, particularly affecting
Asia, with 420,000, 320,000, 131,000, and 101,000 cases reported in the Philippines, Vietnam,
Malaysia, and Bangladesh, respectively [2]. The previous molecular epidemiological studies
in Vietnam described an increasing in the dengue incidence associated with the changes
in DENV-2 genotypes [5]. Recently, the 2017 outbreak was shown to be caused by local
DENV-1 strains [7]. To date, the DENV characteristics responsible for the more recent
outbreak in the 2019–2020 season have not been well elucidated. Therefore, we conducted
a molecular characterization of DENV isolates from patient samples collected between 2019
and 2020 in Hanoi, the capital city of Vietnam located in the northern part of the country,
and nearby provincial cities. We also present clinical data regarding these cases.

2. Materials and Methods

2.1. Patients, Sample Processing, and Laboratory Testing
Clinical samples were collected from patients with suspected dengue infection who
were aged 18 years or older and had a fever (within 5 days of the study). Samples were
collected according to the usual diagnostic procedure of the National Hospital for Tropical
Disease (NHTD), Hanoi, Vietnam. Ethical Approval was obtained from the Ethics Commit-
tee of the NHTD, and all participants gave informed consent. Serum from participating
patients was locally analyzed by Dengue Duo (SD Bioline, Seoul, Republic of Korea) to
detect the NS1 antigen and dengue-specific antibodies (IgM and IgG). RNA extraction and
dengue serotyping were conducted for all NS1-positive serum samples. Viral RNA was
extracted from 140 µL of each serum sample using a QIAamp viral mini kit (Qiagen, Hilden,
Germany), and 5 µL was used to determine the dengue serotype by multiplex real-time
RT-PCR using a Genesig kit (Genesig, Chandler’s Ford, UK).

2.2. Virus Isolation

Thirty microliters of serotyping-positive patient serum was inoculated in C6/36, an
Aedes albopictus cell line, as described previously [8]. A total of 140 µL of culture supernatant
was harvested on days 7 and 14 post-infection, and DENV RNA was extracted and analyzed
using real-time RT-PCR [9].

2.3. Envelope Nested RT-PCR and Whole-Genome RT-PCR

To amplify the DENV envelope region, 5 µL of extracted RNA from patient serum
was primarily reverse transcribed and amplified (1◦ PCR), targeting the first and second
halves of the envelope region using a One-step RT-PCR kit (Qiagen) with primers covering
the envelope region (Table S1). The PCR master mix was prepared according to the
manufacturer’s protocol at a final primer concentration of 0.6 µM. To increase the DNA
concentration, the primary PCR product was subjected to secondary PCR (2◦ PCR) using
GXL polymerase (Takara, Shiga, Japan) and primers (Table S1) that amplified the first and
second halves of the envelope region product.
To amplify the whole DENV genome, 5.5 µL of extracted RNA of each isolate was
used to synthesize cDNA with two primers specific to the first (50 half) and second (30 half)
halves of the genome using SuperScript IV (Invitrogen, Vilnius, Lithuania). The RT reaction
mixture was prepared according to the manufacturer’s protocol. The 50 half and 30 half
Microorganisms 2023, 11, 1267 3 of 16

cDNAs were amplified using corresponding primers [8] and GXL polymerase (Takara,
Shiga, Japan).
The amplified products were examined by gel red staining (Biotium, Fremont, CA,
USA) and agarose gel electrophoresis at 100 volts in TBE buffer. The target band was puri-
fied using Nucleospin kit (MACHEREY-NAGEL, Düren, Germany). DNA concentration
was quantified using either a Nanodrop (Thermo Fisher Scientific, Waltham, MA, USA) or
Qubit system (Invitrogen, Eugene, OR, USA).

2.4. Envelope and Whole-Genome Sequencing

The nucleotide sequence of the genome region encoding the envelope was determined
by Sanger sequencing using an ABI sequencer. The sequencing reactions were prepared
using an ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction kit, version 3.1
(Applied Biosystems, Vilnius, Lithuania). Briefly, 2 µL of DNA template was mixed with
2 µL of reaction mix, 1 µL of 5× sequencing buffer, 1.6 µL of specific primer (Table S1),
and 3.4 µL of nuclease-free water. The amplification cycle conditions were programmed
according to the manufacturer’s protocol.
The whole genome sequences of DENV isolates obtained in the present study were
determined by next-generation sequencing (NGS) on a Miseq platform (Illumina, San
Diego, CA, USA). Preparation of the NGS library using NexteraXT (Illumina, San Diego,
CA, USA) was described previously [8]. The FASTQ results were examined using CLC
Genomics Workbench software, version 21 (CLC Bio, QIAGEN, Valencia, CA, USA). Whole-
genome assembly was conducted using Map read reference command (DENV-1 Mochizuki
AB074760.1 and DENV-2 16681 NC_001474.2 were used as reference strains).
All newly obtained sequences of the envelope and the whole genome were de-
posited in GenBank with accession numbers OQ832560-OQ832594 and OQ832609-
OQ832627, respectively.

2.5. Phylogenetic and Genetic Diversity Analyses

DENV sequences analyzed in the present study were retrieved by February 2023
from GenBank and The Bacterial and Viral Bioinformatics Resource Center (BV-BRC)
(https://www.bv-brc.org/ (accessed on 15 February 2023)) [10] and combined with the
new Vietnam sequences (Table S2). Alignment and translation were conducted in AliView
v1.26 [11]. The maximum-likelihood (ML) tree and model selection were estimated in
IQ-TREE for DENV-1 and DENV-2 using the envelope region [12]. The molecular clock
phylogeny of DENV-2 cosmopolitan based on the envelope region was estimated using
BEAST, v1.10 [13]. The temporal signal dataset was examined by root-to-tip using Tempest
and showed R2 of 0.87 (Figure S1) [14]. Triplicate BEAST runs using settings for the SRD06
and Skygrid coalescent model with either a strict clock or uncorrelated clock model were
employed for >50 million Markov Chain Monte Carlo cycles with 5 million cycle burn-ins,
and marginal-likelihood estimation was performed using path sampling and stepping stone
sampling analyses, which suggested that the strict clock was the best fit for this analysis
(Table S3) [15,16]. The run convergence was accessed in Tracer, v1.7.1 [17]. Triplicate
runs obtaining an effective sample size of >200 were combined in Logcombiner, and the
maximum clade credibility (MCC) phylogeny was generated using TreeAnnotator and
visualized in Figtree, v1.4.4.
To analyze amino acid polymorphism in Hanoi DENVs, the coding sequences were
compared to previous Vietnam sequences available in GenBank. The amino acid position
was annotated corresponding to the reference viruses: NC_001477.1, NC_001274.2, and
GQ398263 for DENV-1 genotype I, DENV-2 genotype Asian-I, and DENV-2 genotype
cosmopolitan, respectively.
Microorganisms 2023, 11, x FOR PEER REVIEW 4 of 17

Microorganisms 2023, 11, 1267

GQ398263 for DENV-1 genotype I, DENV-2 genotype Asian-I, and DENV-2 genotype4 cos-
of 16
mopolitan, respectively.

2.6. Statistical Tests

2.6. Statistical Tests
Differences in categorical and continuous variables were statistically evaluated by
Differences
Fisher’s Exact testinand
categorical and continuous
Mann–Whitney variables were
U test, respectively. statistically
All the statisticalevaluated by
calculations
Fisher’s Exact test and Mann–Whitney U test, respectively. All the statistical
were performed by GraphPad Prism (GraphPad Prism Software, Boston, MA, USA). calculations
were performed by GraphPad Prism (GraphPad Prism Software, Boston, MA, USA).
3. Results
3. Results
3.1.
3.1. DENVs
DENVs in in Hanoi,
Hanoi, Vietnam,
Vietnam,in in2019–2020
2019–2020
Between
Between October and December 2019
October and December 2019 and
and September
September and and December
December 2020, 2020, aa total
total of
of
266 DENV clinical suspected samples were collected at the NHTD (2019,
266 DENV clinical suspected samples were collected at the NHTD (2019, n = 30; 2020, n = 30; 2020, n=
103), withwith
n = 103), 201201
patient serum
patient serum samples
samplesfrom
fromHanoi,
Hanoi, the
thecapital
capitalcity,
city,and
and2424from
from nearby
nearby
provinces
provinces (Hoa
(Hoa Bihn,
Bihn, Phu
Phu Tho,
Tho, Vinh
Vinh Phuc,
Phuc, Bac
Bac Ninh,
Ninh, Hung
Hung Yen,
Yen, Hai
Hai Dung,
Dung, HaHa Nam,
Nam, Thai
Thai
Binh,
Binh, Bac
Bac Giang,
Giang, Bac
Bac Kan,
Kan, Son
Son La,
La, and
and Thanh
Thanh Hoa)
Hoa) in northern
northern Vietnam
Vietnam (Figure
(Figure 1); no
data were available
available for
for 41
41 serum
serum samples.
samples. OfOf these
these samples,
samples, 133133 were
were DENV
DENV NS1–positive
NS1–positive
(49.61%),
(49.61%), 7 were DENV IgM–positive (2.71%),
IgM–positive (2.71%), and 11 were DENV IgG-positive samples
(4.26%). The DENV-positivity
DENV-positivityrate ratewas
was52.47%
52.47%and and20.83%
20.83%for forHanoi
Hanoi city and the provin-
city and the provincial
cial cities
cities (Hai(Hai Duong,
Duong, HoaHoaBihn,Bihn,
PhuPhu
Tho,Tho,
and and
ThanhThanh
Hoa),Hoa), respectively.
respectively.

A B

China

BK

Myanmar

PT VP BG
SL
◉ BN
HN HD
Laos HB
◉ HY ◉
◉ HA TB
Thailand
TH
Vietnam ◉
Cambodia

DENV positive rate

◼ Hanoi city 52.47%
◼ Provincial cities 20.83%
Malaysia ◉ City with DENV cases

Figure
Figure 1. Map
Map ofof Vietnam
Vietnam showing
showing sample
sample sites
sites and
and DENV-positivity
DENV-positivity rates.
rates. (A)
(A) Vietnam
Vietnam isis high-
high-
lighted in blue.
lighted in blue. (B)
(B)Map
Mapofofnorthern
northern Vietnam;
Vietnam; Hanoi
Hanoi (HN)
(HN) is highlighted
is highlighted in blue.
in blue. The provincial
The provincial cities
cities are highlighted
are highlighted in blue
in light light and
blueshown
and shown as follows:
as follows: Hoa Bihn
Hoa Bihn (HB),(HB), Phu(PT),
Phu Tho Tho (PT),
Vinh Vinh
Phuc Phuc
(VP),
(VP), Bac Ninh (BN), Hung Yen (HY), Hai Dung (HD), Ha Nam (HA), Thai Binh (TB), Bac Giang
Bac Ninh (BN), Hung Yen (HY), Hai Dung (HD), Ha Nam (HA), Thai Binh (TB), Bac Giang (BG), Bac
(BG), Bac Kan (BK), Son La (SL), and Thanh Hoa (TH). Cities with DENV cases are marked on the
Kan (BK), Son La (SL), and Thanh Hoa (TH). Cities with DENV cases are marked on the map with a
map with a red dot.
red dot.
Among the 133 DENV-positive patients, case records were available for some, as de-
Among the 133 DENV-positive patients, case records were available for some, as
scribed in Table 1.
described in Table 1.
Table
Table 1.
1. Demographic
Demographic and
and clinical
clinical characteristics
characteristics of
of DENV
DENV patients.
patients.
Total DENV (n = 133) DENV-1 (n = 22) DENV-2 (n = 64)
Total DENV (n = 133) DENV-1 (n = 22) DENV-2 (n = 64) p-Value
Frequency Percentage Frequency Percentage Frequency Percentage p-Value
Frequency Percentage Frequency Percentage Frequency Percentage
Sex
Sex
Male 55 41.35 11 50.00 28 43.75 0.6290 b
Male 55 41.35 11 50.00 28 43.75 0.6290 b b
Female 51 38.35 8 36.36 22 34.38 >0.9999
Female 51 38.35 8 36.36 22 34.38 >0.9999 b
No
No data
data 27
27 20.30
20.30 33 13.64
13.64 14 14 21.88
21.88 0.5413
0.5413 b b
Microorganisms 2023, 11, 1267 5 of 16

Table 1. Cont.

Total DENV (n = 133) DENV-1 (n = 22) DENV-2 (n = 64)

p-Value
Frequency Percentage Frequency Percentage Frequency Percentage
Age
18–29 73 54.89 14 63.64 31 48.44 0.3225 b
30–49 41 30.83 8 36.36 23 35.94 >0.9999 b
>50 13 9.77 0 0.00 8 12.50 0.1071 b
N/A 6 4.51 0 0.00 2 3.13 >0.9999 b
Symptoms a
Fever 99 98.02 17 94.44 48 97.96 0.4681 b
Fatigue 91 88.35 17 94.44 43 87.76 0.6645 b
Muscle pain 55 53.40 9 50.00 21 42.86 0.7823 b
Joint pain 26 25.24 5 27.78 11 22.45 0.7488 b
Skin rash 7 6.80 2 11.11 3 6.12 0.6051 b
Headache 6 5.83 2 11.11 1 2.04 0.1735 b
Tourniquet 5 4.85 1 5.56 3 6.12 >0.9999 b
Abdomen pain 4 3.88 2 11.11 2 4.08 0.2909 b
Joint swelling 3 2.91 1 5.56 1 2.04 0.4681 b
Vomit 2 1.94 0 0.00 2 4.08 >0.9999 b
Other (gingival bleeding, ascites,
3 2.91 0 0.00 3 6.12 0.5581 b
and reduction in urination)
Chemical a Median IQR Median IQR Median IQR
115.50–
Platelets (103 /µL) 158 126–185 131.00 162 129–183 0.2470 c
194.75
White blood cells (103 /µL) 4.6 3–5.9 3.8 2.98–5.98 4.9 3.15–6.3 0.3337 c
Hemoglobin (g/L)
Male 148 141–155.5 146 140–155 147 141–153 0.9431 c
Female 131 125–141.25 124 111–145 131 126–134.25 0.1245 c
Hematocrit (%)
Male 43.3 41.4–45.3 43 41.6–44.9 42.5 40.8–45.3 0.9557 c
36.75–
Female 38.35 36.6 34–43.8 38.45 36.95–39.9 0.2858 c
41.35
a Data available for only 18 DENV-1 and 49 DENV-2 cases. b Fisher’s exact test. c Mann–Whitney test.

Among the 88 DENV serotyped cases, 22 (25%), 64 (72.73%), and 2 (2.27%) were
identified as DENV-1, DENV-2, and DENV-4, respectively. DENV-2 was the predominant
serotype in 2019 and 2020. The demographic and clinical characteristics of DENV-1 and
DENV-2 cases are summarized in Table 1, and there was no significant difference in
demographic and clinical laboratory data between DENV-1 and DENV-2 cases. Molecular
characterization was performed for 36 cases, consisting of 13 DENV-1 and 23 DENV-2
strains, as presented in Table 2. Unfortunately, the DENV-4 sequence was unobtainable
due to the low viral load.

Table 2. DENV strains characterized in the present study.

Accession Number
Resident Anti- Anti-
Strain Country Collection Date NS1 Ag Serotype Genotype Full
Area IgM IgG Envelope
Genome
DENVN19_075 ND Vietnam November 2019 POS NEG NEG DENV-1 I OQ832560 OQ832609
DENVN19_080 Hanoi Vietnam December 2019 POS NEG NEG DENV-1 I OQ832561 OQ832610
DENVN19_129 ND Vietnam October 2019 POS NEG NEG DENV-1 I ud OQ832611
DENVN19_137 ND Vietnam October 2019 POS NEG NEG DENV-1 I OQ832562 OQ832612
DENVN20_020 Phu Tho Vietnam September 2020 POS POS NEG DENV-1 I OQ832563 ud
Microorganisms 2023, 11, 1267 6 of 16

Table 2. Cont.

Accession Number
Resident Anti- Anti-
Strain Country Collection Date NS1 Ag Serotype Genotype Full
Area IgM IgG Envelope
Genome
DENVN20_032 Hanoi Vietnam September 2020 POS NEG NEG DENV-1 I OQ832564 ud
DENVN20_059 Thanh Hoa Vietnam September 2020 POS NEG NEG DENV-1 I OQ832565 ud
DENVN20_066 Hanoi Vietnam October 2020 POS NEG NEG DENV-1 I OQ832566 ud
DENVN20_111 Hanoi Vietnam September 2020 POS NEG NEG DENV-1 I OQ832567 OQ832613
DENVN20_112 Hanoi Vietnam September 2020 POS NEG NEG DENV-1 I OQ832568 OQ832614
DENVN20_120 Hanoi Vietnam September 2020 POS NEG NEG DENV-1 I OQ832569 OQ832615
DENVN20_124 Hanoi Vietnam September 2020 POS NEG NEG DENV-1 I OQ832570 ud
DENVN20_226 Hanoi Vietnam October 2020 POS NEG NEG DENV-1 I OQ832571 ud
DENVN19_004 ND Vietnam October 2019 POS NEG NEG DENV-2 Cosmopolitan OQ832572 OQ832616
DENVN19_006 ND Vietnam October 2019 POS NEG NEG DENV-2 Cosmopolitan OQ832573 ud
DENVN19_010 ND Vietnam October 2019 POS NEG NEG DENV-2 Asian-I OQ832574 OQ832617
DENVN19_011 ND Vietnam October 2019 POS NEG NEG DENV-2 Cosmopolitan OQ832575 OQ832618
DENVN19_013 ND Vietnam October 2019 POS NEG NEG DENV-2 Cosmopolitan OQ832576 OQ832619
DENVN19_015 ND Vietnam October 2019 POS NEG NEG DENV-2 Cosmopolitan OQ832577 OQ832620
DENVN19_078 ND Vietnam December 2019 POS NEG NEG DENV-2 Cosmopolitan OQ832578 OQ832621
DENVN19_089 Hanoi Vietnam December 2019 POS NEG NEG DENV-2 Cosmopolitan OQ832579 OQ832622
DENVN19_140 ND Vietnam October 2019 POS NEG NEG DENV-2 Asian-I OQ832580 ud
DENVN19_142 ND Vietnam October 2019 POS NEG NEG DENV-2 Cosmopolitan OQ832581 ud
DENVN19_143 ND Vietnam October 2019 POS NEG NEG DENV-2 Asian-I OQ832582 OQ832623
DENVN19_144 ND Vietnam October 2019 POS NEG NEG DENV-2 Cosmopolitan OQ832583 ud
DENVN20_019 Hanoi Vietnam September 2020 POS NEG NEG DENV-2 Cosmopolitan OQ832584 ud
DENVN20_021 Hanoi Vietnam September 2020 POS NEG NEG DENV-2 Cosmopolitan OQ832585 ud
DENVN20_049 Hanoi Vietnam September 2020 POS NEG NEG DENV-2 Cosmopolitan OQ832586 ud
DENVN20_074 Hanoi Vietnam October 2020 POS NEG NEG DENV-2 Cosmopolitan OQ832587 ud
DENVN20_106 Hanoi Vietnam September 2020 POS NEG NEG DENV-2 Cosmopolitan OQ832588 ud
DENVN20_107 Hanoi Vietnam September 2020 POS NEG NEG DENV-2 Cosmopolitan OQ832589 OQ832624
DENVN20_113 Hanoi Vietnam September 2020 POS NEG NEG DENV-2 Cosmopolitan OQ832590 OQ832625
DENVN20_118 Hanoi Vietnam September 2020 POS NEG NEG DENV-2 Asian-I OQ832591 OQ832626
DENVN20_127 Hanoi Vietnam September 2020 POS NEG NEG DENV-2 Asian-I OQ832592 OQ832627
DENVN20_210 Hanoi Vietnam October 2020 POS NEG NEG DENV-2 Cosmopolitan OQ832593 ud
DENVN20_220 Hanoi Vietnam October 2020 POS NEG NEG DENV-2 Cosmopolitan OQ832594 ud
ND: no data; POS: positive; NEG: negative; ud: undetermined.

3.2. Persistence of DENV-1 Genotype I in Vietnam

The ML tree of the envelope gene region (1485 bp) of the newly obtained sequences
was reconstructed together with sequences from GenBank, including DENV-1 genotype
references (I–V) and the related sequences from BLASTN searches (Figure 2). All DENV-1
isolates belonged to genotype I and were separated into two clades. First, two DENV-1
isolates (DENVN20_120 and DENVN20_20) were the most closely related to the Vietnam
strains collected in 2022 and clustered with Vietnam strains circulating in 2015–2017. Sec-
ond, the remaining DENV-1 isolates formed the majority (DENVN19_129, DENVN19_137,
DENVN19_075, DENVN19_080, DENVN20_124, DENVN20_032, DENVN20_059, DENVN
20_066, and DENVN20_226) of those related to the 2017 strains collected in China and
Vietnam.

3.3. Co-Circulation of the Asian-I and Cosmopolitan Genotype of DENV-2 in Vietnam

The ML tree of the DENV-2 envelope sequences (Figure 3) consisted of the Hanoi 2019–
2020 strains reported in the present study, their related sequences obtained by BLASTN
searches, other Vietnam strains available in the database, and the genotype representative
strains. Five distinct genotypes were separated with branch support (bootstrap 100%). The
Hanoi DENV-2 isolates were classified into two genotypes. First, five isolates, including
DENVN19_010, DENVN19_140, DENVN19_143, DENVN20_118, and DENVN20_127,
belonged to the Asian-I genotype and clustered closely with recently reported viruses
isolated from Vietnam in 2017–2022, China in 2017–2019, and Cambodia in 2019. Second,
the vast majority (18 strains shown in Table 1) were characterized as the cosmopolitan
genotype. Of these cosmopolitan isolates, DENVN19_015 and DENVN19_144, which were
collected in 2019, clustered to a small clade with the most-related strain from Thailand
collected in 2016 and 2018, whereas the remaining 16 strains (2019–2020) formed a clade
together with other Vietnam strains circulating in 2018–2021 and Australia strains collected
in 2019.
Microorganisms 2023,
Microorganisms 11,11,1267
2023, x FOR PEER REVIEW 7 of 16
7 of 17

Figure 2.
Figure 2. Maximum-likelihood
Maximum-likelihood tree treeofofDENV-1
DENV-1isolates from
isolates Vietnam
from based
Vietnam on the
based envelope
on the gene.gene.
envelope
The best nucleotide substitution model was TIM2 + F + G4. The Vietnam strains sequencedthe
The best nucleotide substitution model was TIM2 + F + G4. The Vietnam strains sequenced in in the
present study are labeled in red. Of them, the sequences obtained from the whole-genome analysis
present study are labeled in red. Of them, the sequences obtained from the whole-genome analysis
are marked with an asterisk. Public Vietnam sequences from GenBank are labeled in blue. Geno-
are marked
types with an
of DENV-1 areasterisk.
indicatedPublic
to theVietnam sequences
right. Numbers from GenBank
on branches are
indicate labeled in
bootstrap blue. Genotypes
support values
of DENV-1
(>75%). are indicated to the right. Numbers on branches indicate bootstrap support values (>75%).

Since we observed
3.3. Co-Circulation that the
of the Asian-I and DENV-2
Cosmopolitancosmopolitan
Genotype of genotype
DENV-2 in predominated
Vietnam in the
Hanoi epidemic of 2019–2020, we collected public sequences to determine the historical
The ML tree of the DENV-2 envelope sequences (Figure 3) consisted of the Hanoi
distribution of DENV-2 genotypes in Vietnam from 1988–2022 (Figure 4). The results
2019–2020 strains reported in the present study, their related sequences obtained by
indicated that there was no circulation of the Asian-American genotype after the mid-
BLASTN searches, other Vietnam strains available in the database, and the genotype rep-
dle of the 2000s,
resentative strains.butFiveAsian-I
distinctwas the major
genotypes were genotype overbranch
separated with most of the 2005–2013
support (bootstrapand
2016–2019
100%). The Hanoi DENV-2 isolates were classified into two genotypes. First, fiveenvelope
period. Regarding the phylogeny of DENV-2 (Figure 3), Asian-I isolates, se-
quences sampled between 2003 and 2022 formed
including DENVN19_010, DENVN19_140, DENVN19_143, DENVN20_118,clusters and descended from earlier
andViet-
nam
DENVN20_127, belonged to the Asian-I genotype and clustered closely with recently re- S2.
strains, which is consistent with the larger dataset analysis summarized in Figure
This genetic
ported virusesrelationship
isolated from suggested
Vietnam in that the Vietnam
2017–2022, ChinaAsian-I lineageand
in 2017–2019, was localizedinand
Cambodia
maintained until the present. On the other hand, the cosmopolitan
2019. Second, the vast majority (18 strains shown in Table 1) were characterized genotype hadasmultiple
the
introductions. It was detected sporadically in 2006–2007,
cosmopolitan genotype. Of these cosmopolitan isolates, DENVN19_015 2009, and 2011 (Figure 4). Notably,
and
the cosmopolitanwhich
DENVN19_144, genotype
weredominated
collected infor a short
2019, time in
clustered to 2014 andclade
a small 2015,with
at 100% and 84%,
the most-
respectively.
related strainThe
fromcosmopolitan genotype
Thailand collected recently
in 2016 re-emerged
and 2018, whereasbetween 2018 and
the remaining 2022 and
16 strains
exhibited
(2019–2020)a trend
formed ofaincreasing proportion
clade together with otherduring thisstrains
Vietnam period, at 5, 17, 79,
circulating 50, and 89%.
in 2018–2021
Based on our results,
and Australia we observed
strains collected the cosmopolitan genotype at 75% and 83% of DENV-2
in 2019.
samples in 2019 and 2020, respectively.
Microorganisms
Microorganisms2023,
2023,11,
11,x 1267
FOR PEER REVIEW 8 8ofof1716

Figure
Figure3.3.Maximum-likelihood
Maximum-likelihoodtree treeofofDENV-2
DENV-2isolates
isolatesfrom
fromVietnam
Vietnambased
basedononthe
theenvelope
envelope gene.
gene.
The
The best nucleotide substitution model was TIM2 + F + I + G4. The Vietnam strains sequenced inthe
best nucleotide substitution model was TIM2 + F + I + G4. The Vietnam strains sequenced in the
present
presentstudy
studyare
arelabeled
labeledininred.
red.Of
Ofthem,
them,the
thesequences
sequencesobtained
obtainedfrom
fromwhole
wholegenome
genomeanalysis
analysisare
are
marked with an asterisk. Public Vietnam sequences from GenBank are labeled in blue. Genotypes
marked with an asterisk. Public Vietnam sequences from GenBank are labeled in blue. Genotypes of
of DENV-2 are indicated to the right. Numbers on branches indicate bootstrap support values
DENV-2 are indicated to the right. Numbers on branches indicate bootstrap support values (>75%).
(>75%).
 politan genotype dominated for a short time in 2014 and 2015, at 100% and 84%, r
tively. The cosmopolitan genotype recently re-emerged between 2018 and 2022 and
ited a trend of increasing proportion during this period, at 5, 17, 79, 50, and 89%.
on our results, we observed the cosmopolitan genotype at 75% and 83%9of
Microorganisms 2023, 11, 1267 DENV-
of 16
ples in 2019 and 2020, respectively.

Figure Figure
4. Proportion of of
4. Proportion the
theDENV-2 genotype
DENV-2 genotype in Vietnam
in Vietnam from
from 1988 1988
to 2022 to 2022
based based on seq
on sequences
available in GenBank.
available in GenBank.

3.4. Emergence of DENV-2 Cosmopolitan Lineage C (Asian-Pacific) in Vietnam

3.4. Emergence of DENV-2
The time Cosmopolitan
to the most Lineage
recent common C (tMRCA)
ancestor (Asian-Pacific) in Vietnam
of the DENV-2 genotype
cosmopolitan
The time to the was estimated
most recentusing BEAST,ancestor
common and the result is summarized
(tMRCA) in an MCCgenotyp
of the DENV-2
tree in Figure 5. The dataset consisted of the reference strains representing the DENV-2
mopolitan was estimated
cosmopolitan using BEAST,
lineages previously and[8,18],
described the all
result is summarized
available in an MCC
Vietnam and related
Figurestrains
5. Thefromdataset consisted
the database, andofstrains
the reference strains
reported during representing
2015–2022. Three the DENV-2 c
lineages
separated with a posterior of 1, including lineage A (African),
politan lineages previously described [8,18], all available Vietnam and related lineage B (Indian), and strain
lineage C (Asian-Pacific). Within lineage C (Asian-Pacific), six phylogenetic clades
the database, and were
(Clades i–vi) strains reported
observed during
during 2015–2022.
the 2014–2022 Three
period. The lineages separated
basal strains of each with
terior of 1, including
clade lineage viruses
were cosmopolitan A (African), lineageprior
that circulated B (Indian), and in
to mid-2010 lineage C (Asian-P
countries in
Withinmaritime
lineageSoutheast Asia, including
C (Asian-Pacific), six Singapore,
phylogenetic Indonesia,
cladesand Malaysia,
(Clades which
i–vi) werewere
observe
cosmopolitan virus hotspots.
ing the 2014–2022 period. The basal strains of each clade were cosmopolitan viruse
In Figure 5, the Vietnam cosmopolitan viruses formed seven clusters. Cluster 1
circulated prior
strains fromto2006–2011
mid-2010 fellininto
countries
lineage Cinfrom
maritime
China inSoutheast Asia,
2010, and the including
tMRCA was Sing
Indonesia, and Malaysia, which were cosmopolitan virus hotspots.
estimated at 2004.2 (2003.3–2005.1). Cluster 2 strains from 2014–2015 were in lineage
B and related to the Indian strain from 2014. The tMRCA was 2012.3 (2011.5–2013.2).
Notably, recent Vietnam strains collected during 2018–2022 classified in lineage C
separated in clusters 3–7. Cluster 3 was related to the Indonesia strains from 2016, and
the tMRCA was estimated at 2016.0 (2015.2–2016.8). The vast majority of our Hanoi
strains from 2019–2020 were in this cluster together with the Ho Chi Minh City strain
collected in 2018–2020. Furthermore, a Vietnam strain collected in 2020 (OQ028232) in
Cluster 3 was most closely related to Australia 2019 strains (MN982899-MN982901).
Viruses of Clusters 4, 5, and 6 were most closely related to viruses from China, Thailand,
and Cambodia that circulated in 2018–2020, and the tMRCA of these viruses was
2016.7 (2015.8–2017.7), 2017.0 (2016.4–2017.6), and 2018.8 (2018.6–2019.1), respectively.
Interestingly, a minor group of Hanoi isolates (DENVN19_015 and DENVN19_144)
belonged to Cluster 6 and was distinctly separated from the majority of Hanoi viruses
(cluster 3). The ancestral strain of these isolates was closely related to Thailand strains
(MN955677 and LC410190). Five Vietnam strains collected in 2022 were in Cluster 7 in
which the tMRCA was dated at 2019.8 (2018.7–2020.9). These strains were related to
the Indonesia strains from 2016.
Microorganisms 2023, 11,
Microorganisms 1267
2023, 11, x FOR PEER REVIEW 10 of 1710 of 16

Figure
Figure 5. Maximumclade
5. Maximum cladecredibility
credibility tree
treeofofDENV-2
DENV-2 genotype cosmopolitan
genotype basedbased
cosmopolitan on theon
envelope
the envelope
gene.
gene. TheThe Vietnamsequences
Vietnam sequences obtained
obtained ininthe thepresent
presentstudy andand
study from GenBank
from are labeled
GenBank in red in red
are labeled
and blue, respectively. The grey and dotted grey brackets indicate the lineage and clade, respectively.
The yellow bracket indicates Vietnam clusters with the most recent common ancestor (tMRCA) and a
95% highest posterior density interval (HPD). The numbers of posterior probability (PP) support are
shown adjacent to the key nodes. The branch tip color corresponds to the location indicated. The
timescale in years is shown on the x-axis at the bottom.
Microorganisms 2023, 11, 1267 11 of 16

3.5. Amino Acid Polymorphisms within Genotypes of Hanoi DENV Strains

The whole genomes of DENV isolates obtained in the present study were analyzed for
amino acid polymorphisms and compared to the previous Vietnam strains (Figure 6). The
envelope sequences analyzed above showed complete consistency with the corresponding
region in the whole genome sequences for each sample. Among seven strains of DENV-1
genotype I, a total of 22 amino acid differences were observed. DENVN20_111 had the
most newly observed mutations, with 4 amino acid differences compared to other strains.
For DENV-2 genotype Asian-I strains, 22 different amino acids were observed along the
entire coding sequence. The major clade strains had mutations similar to those of the
Vietnam strains reported in 2017–2018. Among DENV-2 genotype cosmopolitan viruses,
35 different amino acid substitutions located in both structural and nonstructural genes
were observed. The DENVN19_015 isolate, classified in Cluster 6, showed six newly
observed mutations, whereas the remaining isolates (DENVN19_004, DENVN19_011,
DENVN19_013,
Microorganisms 2023, 11, x FOR PEER REVIEW DENVN19_078, DENVN19_089, DENVN20_107, and DENVN20_113) that
12 of 17
were grouped in Cluster 3 shared the seven specific mutations of NS1-146I, NS1-178L,
NS2A-137I, NS2A-171I, NS3-31F, NS3-519V, and NS5-648E.

A. DENV-1 genotype I

Collection preM ENV NS1 NS2A NS3 NS4A NS5

Strain Clade
Year 127 136 52 320 409 485 173 213 246 38 118 143 171 323 76 97 65 199 200 642 647 833
NC_001477.1 1974 - A I N V A V D A I A N H I V R M F K H V K E
Vietnam (n=66) 2017 - A I/M N/D V A V D A/T I/V A/V N H/Y V/I V/M K/R M/V F/L K/R H/Y V/A E/G G
DENVN19_129 2019 major A I N V A V D T V A N H I M R M L R H A G G
DENVN19_075 2019 major A I N V A V D T V A N H I M R M L R H A G G
DENVN19_080 2019 major A I N V A V G T V A N H I M R M L R H A G G
DENVN20_112 2020 major A I N V A V G T V A S H I M R M L R H A G G
DENVN19_137 2019 major V I N V V V D T V A N H I M K M L R H A G E
DENVN20_111 2020 major V I N I V V D T V A N Y I M R M L R H A G E
DENVN20_120 2020 minor A M D V A I D A I V N H V V K V F K Y V E G

B. DENV-2 genotype Asian-I

Collection Capsid ENV NS1 NS2A NS3 NS5

Strain Clade
Year 72 160 347 129 177 202 246 351 36 104 109 167 577 590 5 76 176 286 400 628 631 898
NC_001474.2 1964 - I K V H V I N T V T V V V K I K T Q T V S V
Vietnam (130) 2006–2008 - I K/Q/M V H/Y A I/M N T V/A T V V V/M K I/M K T Q T V N/S V
Vietnam (216) 2017–2020 - V/I M A/V Y V/A/T I N/S S/T V T V V V K I K T E T I/V N V
DENVN19_143 2019 minor V M A Y T I N S V A A A V R I K I E I V N V
DENVN19_010 2019 major V M A Y A I S S A T V V V K I R T E T I N V
DENVN20_118 2020 major V M A Y A M S S V T V V M K I K T E T I S A
DENVN20_127 2020 major V M A Y A M S S V T V V M K I K T E T I S V

C. DENV-2 genotype Cosmopolitan

Collection Capsid PreM Env NS1 NS2A NS2B NS3 NS4A NS5
Strain Cluster
Year 14 63 104 15 173 345 484 80 96 109 146 178 251 29 119 137 171 189 21 59 99 15 29 31 395 519 89 271 388 637 648 676 698 865 898
GQ398263.1 1975 - N A M R A R V S I T T L V T T V I A L V I K G L I I I I K V A S R T V
Vietnam (n=3) 2006 1 N A I R A R V S I T T F V T T V T A L V I K G L I I I T K V A S R T V
Vietnam (n=6) 2018 5 N A I R A R V T I T T S V T T V A T F I I R G L T I V A K A V N K T V
Vietnam (n=4) 2019 4, 5 N A I R A R V T I T T S V T T V A T F I I R G L I/T I V A/V K/E A V N R/K T V
Vietnam (n=3) 2020 3, 4 N A I R A R V/I S/T I T T/I S/L V T T V/I A/I T/A F/L V/I I K/R G L/F I I/V I/V T/V K V/A V/E S/N R T V
DENVN19_004 2019 3 N A I R A R I S I T I L V T T I I A L V I K G F I V I T E V E S R T V
DENVN19_011 2019 3 S A I K A K I S I T I L V T T I I A L V I K G F I V I T K V E S R T V
DENVN19_013 2019 3 N A I R A R I S I T I L V T N I I A L V I K G F I V I T K V E S R T V
DENVN19_078 2019 3 N T I R A K I S I T I L V T T I I A L V I K G F I V I T K V E S R I V
DENVN19_089 2019 3 N A I R A R I S V T I L V M T I I A L V I K G F T V I T K V E S R T V
DENVN20_107 2020 3 N A I R A R V S I T I L V T T I I A L V I K G F I V I T K V E S R T V
DENVN20_113 2020 3 N A I R A R V S I T I L A T T I I A L V V K G F I V I T K V E S R T V
DENVN19_015 2019 6 N T V R V R V T I I T S V T T V A T F I I R R L I I V A K A V N K T A

Figure 6. Amino acid polymorphisms within genotypes of Hanoi DENV strains compared to the
Figure 6. Amino acid polymorphisms within genotypes of Hanoi DENV strains compared to the
previous Vietnam strains. The codon numbering with respect to the reference viruses of
previous Vietnam strains. The codon numbering with respect to the reference viruses of NC_001477.1,
NC_001477.1, NC_001274.2, and GQ398263.1 for DENV-1 genotype I, DENV-2 genotype Asian-I,
NC_001274.2,
and DENV-2 and GQ398263.1
genotype for DENV-1
cosmopolitan, genotype
respectively. TheI, variations
DENV-2 genotype Asian-I,inand
newly observed the DENV-2
present
genotype
study andcosmopolitan, respectively.
the amino acids specific to The variations
certain newlyare
viral clusters observed in blue
labeled in the present
and red,study and the
respectively.
amino acids specific
The number to certain
of previous viralstrains
Vietnam clusters
is are labeledininthe
indicated blue and red, respectively. The number of
parenthesis.
previous Vietnam strains is indicated in the parenthesis.
4. Discussion
4. Discussion
In 2019, more than 300,000 dengue cases were reported in Vietnam, an approximately
In 2019,increase
three-fold more than 300,000
from dengue year
the previous cases[19].
wereInreported in Vietnam,
the present an approximately
study, DENV patients in
three-fold increase from the previous year [19]. In the present study, DENV
Hanoi city and nearby provinces in the northern part of Vietnam during October-Decem- patients in
Hanoi city and nearby provinces in the northern part of Vietnam during October-December
ber 2019 and September-October 2020 were investigated. The DENV NS1-positivity rate
2019
wasand September-October
higher 2020most
in urban areas, with wereDENV
investigated. The DENV
cases detected NS1-positivity
in Hanoi rate
involving no was
travel
higher in urban
history, areas,
and four with
cases most DENV
occurred cases
in rural detected
areas. Thesein positivity
Hanoi involving no travelthat
rates indicate history,
the
and four cases occurred in rural areas. These positivity rates indicate that the transmission
transmission significantly occurred in the more densely populated areas [20]. Most of the
DENV patients in the present study had primary infection with mild illness showing fe-
ver, fatigue, and muscle pain as the typical symptoms. A cohort study of hospitalized
adult patients in northern Vietnam during 2016–2019 reported that dengue with warning
signs in primary infection were found in 33% of patients in the 2017–2018 season and in
Microorganisms 2023, 11, 1267 12 of 16

significantly occurred in the more densely populated areas [20]. Most of the DENV patients
in the present study had primary infection with mild illness showing fever, fatigue, and
muscle pain as the typical symptoms. A cohort study of hospitalized adult patients in
northern Vietnam during 2016–2019 reported that dengue with warning signs in primary
infection were found in 33% of patients in the 2017–2018 season and in 17% in the 2018–2019
season. Dengue with warning signs was found more often in secondary infection, at 40%
in the 2017–2018 season and at 21% in the 2018–2019 season, while there were few cases of
severe dengue [21]. DENV-1 was reported as the primary serotype responsible for the 2017
epidemic [7,22], whereas DENV-2 was the predominant serotype in the 2019–2020 season.
These results suggested that the change in serotype influenced the clinical profile seen in
the present study.
DENV-1 and DENV-2 were the major serotypes, and very few DENV-4 could be
detected in the present study. DENV-2 was the predominant serotype both in 2019 and
2020. In the 2017 epidemic, DENV-1 was the dominant serotype in locations in northern
Vietnam such as Hanoi, Ha Nam, and Hai Duong. Subsequently, DENV-1 decreased
gradually from 2017 to 2019 perhaps due to the presence of serotype-specific immunity
in the human population [7,21–23]. Notably, the serotype shift from DENV-1 to DENV-2
noted in our study occurred primarily in northern Vietnam in 2019–2020. However, other
regions, such as central Vietnam, had a different predominant type during the period
from December 2018 to February 2019, with mostly DENV-4 and a small proportion of
DENV-2 [24]. Serotype replacement occurred in southern Vietnam as DENV-2 observed
between 2003 and 2006 [5] switched to DENV-1 between 2006 and 2008 [20], whereas in
northern Vietnam, and particularly Hanoi, the proportions of DENV-1 and DENV-2 were
reportedly equal in 2008 [25]. Dengue transmission is high in Ho Chi Minh City and
southern Vietnam, with the annual wave of cases typically peaking in the dry season [26].
Unlike in southern Vietnam, Hanoi has a subtropical climate with four seasons. Dengue
transmission in northern Vietnam driven by DENVs from the southern region is usually
interrupted by a seasonal bottleneck [27]. However, recent changes in climate have altered
the situation. This has led to a greater frequency of spread in the northern region and a
higher incidence of dengue, especially between June and November, when the highest
temperatures of the year occur [28,29]. Thus, DENV surveillance is required routinely to
monitor the new serotype such as DENV-2 in Hanoi through the winter since the serotype
shift would link to an increase in the patient number.
We detected DENV-1 as a minor serotype with a single genotype as all 13 DENV-1
isolates were phylogenetically classified as genotype I. Although these viruses clustered
in two separate clades, they were related and clustered with local strains that were also
collected in 2017, 2019–2020, and 2022. A total of 59,063 dengue fever cases were reported
in the 2017 epidemic in northern Vietnam, which was eight times the number of cases
reported in 2016 that were associated with DENV-1 genotype I [7,30]. Our results showed
that the 2019–2020 DENV-1 genotype I isolates descended from viruses that caused the 2017
epidemic that persisted in this region. Indeed, DENV-1 genotype I has existed in Vietnam
since it was first introduced from Thailand during the late 1980s or early 1990s, and it has
been imported multiple times from Cambodia in the 2000s [20,27]. DENV-1 genotype I
subsequently circulated as the dominant genotype and has now descended into several
local clades associated with the latter outbreaks [7].
Among DENV-2 isolates detected in the present study, the cosmopolitan genotype
was predominant over the Asian-I genotype during 2019–2020. Genotype replacement
has occurred in Vietnam over the last decade. Asian-I, which initially arose as the new
type in 2003, subsequently replaced the Asian-American type, the previous local type,
between 2003 and 2007 [5]. Since then, the Asian-I type has circulated sustainably until
the present [31,32]. However, the co-circulation of Asian-I and cosmopolitan viruses was
observed not only in the present study but also in a previous study involving a DENV-
infected visitor who had a travel background in Vietnam; that study described the trend
in genotype distribution during 2003–2016 [33]. Co-circulation was detected in the single
Microorganisms 2023, 11, 1267 13 of 16

years 2007 and 2011 and continuously between 2013 and 2015. Regarding our DENV-2
phylogenetic tree, Vietnam cosmopolitan viruses were observed in several distinct clades
from 2006–2022. The Vietnam 2006–2011 virus (Cluster 1, which was part of a small cluster
within lineage C (Asian-Pacific)) and the 2014–2015 virus (Cluster 2, which was within
lineage B (Indian)) are both no longer detected. The Vietnam 2018–2022 cosmopolitan
viruses were associated with lineage C (Asian-Pacific). In particular, the viruses of clusters
4–6 are closely related to virus strains from bordering countries, including Cambodia,
Thailand, and China, that were isolated in 2018–2019 and are probable origins. In contrast,
viruses of Clusters 3 and 7 had Indonesia and Singapore strains isolated in 2016 as ancestral
strains, indicating that multiple, independent introductions occurred in Vietnam. In the
case of Hanoi, most of the Hanoi viruses were in Cluster 3 and were very close to the
Ho Chi Minh City strain, the earliest of which was detected in 2018. Moreover, Cluster
3 viruses shared specific amino acid mutations (NS1-146I, NS1-178L, NS2A-137I, NS2A-
171I, NS3-31F, NS3-519V, and NS5-648E). This suggests that the virus that spread across
southern and northern Vietnam and Ho Chi Minh City was likely the origin of the Hanoi
cosmopolitan virus.
Global cosmopolitan isolates sampled from public databases and isolated in different
geographic regions, including Asia, Oceania, Africa, and South America, also fall into lin-
eage C (Asian-Pacific), indicating a global spread with increased DENV cases or outbreaks.
Their emergence in southeast Asia, including Thailand 2016–2017, Cambodia 2019–2020,
and Vietnam 2018–2022, led to co-circulation with Asian-I, the local genotype [8,34]. In
areas of southern Asia, including Nepal 2017, Sri Lanka 2017–2018, Bangladesh 2017–2018,
the Maldives 2017–2019, India 2021, and Pakistan 2022 [35–37], the cosmopolitan lineage
C virus emerged, and this is where the lineage B (Indian) virus had previously circulated.
Furthermore, the cosmopolitan lineage C virus spread for the first time to South America,
with local transmission reported in Peru in 2019 [38] and Brazil in 2022 [39], both of which
were mostly related to the Bangladesh 2017 virus [37]. It is possible that the cosmopolitan
lineage C virus successfully adapted to humans, particularly in transmission fitness, result-
ing in its spread to a wider area and an increase in cases of infection. In the case of Vietnam,
more than 300,000 individuals were affected in 2019. Monitoring DENV lineages could
provide a better assessment of the epidemic risk as well as advice on resource allocation
and guided control actions to minimize transmission intensity.
Multiple DENV clades are often observed in hyperendemic areas. Here, we reported
the prevalent clades circulating in Hanoi and the amino acid polymorphisms. Regarding
the diversity within DENV genotypes, differences at the nucleotide and amino acid levels
were less than 6% and 3%, respectively [40]. DENV has acquired viral fitness or vector
competence, and both these mechanisms are known to be associated with viral turnover
events such as the persistence or replacement of clades/lineages [4]. For instance, the
K160Q/M mutation in the DENV-2 genotype Asian-I viruses that emerged in Vietnam
in 2008–2011 caused higher viremia in patients but increased neutralization sensitivity
in DENV-2 genotype Asian-American [41]. Interestingly, the K160M mutation was still
maintained and detected in the Vietnam Asian-I lineage in the present study. In addition,
we have identified new mutations that were suspected to play certain roles in fitness.
Their phenotypic effects are still unknown and should be investigated further. Several
studies have explained that there was greater replication of the major/dominant clade
virus in native mosquitos, thereby enhancing the local transmission. This has been seen in
the DENV-1 genotype I clade/lineage shift in Thailand and Cambodia and the DENV-2
Asian-American dominant clade replacement in Nicaragua [42–44]. As mentioned above,
the newly introduced cosmopolitan lineage C (Asian-pacific) is invading into new areas
and subsequently co-circulating with the local genotype, such as Asian I, as shown in the
present study. However, the mechanisms of viral evolution are still unclear. Further studies
are required to explore the mechanisms or factors involving viral turnovers.
Microorganisms 2023, 11, 1267 14 of 16

The limitations of this study include the lack of travel history of patients that could
illustrate the virus transmission more clearly. Detailed demographic and clinical laboratory
data were not available for some patients. The available Vietnam DENV sequences in
the database are also limited in some periods. Therefore, more sequence data are critical
to monitor the spread of the virus. In conclusion, our study characterized DENV strains
associated with a large dengue outbreak in 2019–2020 in northern Vietnam and enhanced
our understanding of the recent dynamics of DENV transmission. DENV-1 genotype I and
DENV-2 genotype Asian-I are still maintained, but DENV-2 genotype cosmopolitan has
re-emerged. We demonstrated that the Hanoi cosmopolitan strain was associated with
lineage C (Asian-Pacific) and related to viruses from neighboring countries. In addition,
identifying the dengue genotype and estimating the time the outbreak strain emerged are
important for determining the origin, routes of transmission, and circulation of DENVs as
well as for evaluating vaccine performance and virus control efforts.

Supplementary Materials: The following supporting information can be downloaded at https://

www.mdpi.com/article/10.3390/microorganisms11051267/s1. Table S1: PCR and sequencing primers
targeting the envelope gene region; Table S2: Sequences analyzed in the present study; Table S3:
The model-fit comparison of the log marginal likelihood estimation (MLE) using path sampling (PS)
and stepping-stone sampling (SS); Figure S1: Temporal signal analysis of regression of root-to-tip
divergence against date; Figure S2: Phylogenetic tree of Vietnam strains from 1988 to 2022. The
envelope sequences of 760 Vietnam DENV-2 strains from 1988 to 2022 and DENV-2 genotype reference
strains were constructed for the maximum-likelihood tree under TIM2 + F + I + G4. The DENV-2
genotypes and the Bootstrap (>80%) are indicated at the adjacent branch. The tree branch is colored
corresponding to genotypes. Vietnam taxa from a public database and obtained in the present study
are labeled in blue and red, respectively.
Author Contributions: Conceptualization, H.T.N.N., E.E.N., and A.I.; methodology, J.P.; validation,
H.T.T.V., Q.T.N., A.I., and T.S.; formal analysis, J.P.; investigation, J.P.; resources, H.T.T.V., H.T.V.N.,
H.T.N.N., and B.T.D.; data curation, H.T.T.V., Q.T.N., and E.E.N.; writing—original draft preparation,
J.P.; writing—review and editing, H.T.T.V., A.I., H.I., T.S., and T.N.P.; visualization, J.P.; supervision,
H.T.T.V., E.E.N., A.I., H.I., T.S., and T.N.P.; project administration, H.T.T.V., A.I., H.I., T.S., and T.N.P.;
funding acquisition, E.E.N. and T.S. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was funded by the Japan Agency for Medical Research and Development
under grant numbers 20wm0225010h0101 and 21wm0225010h0102. The APC was funded by the
Research Institute for Microbial Diseases, Osaka University.
Institutional Review Board Statement: Ethical approval was obtained from the Ethics Committee
of the National Hospital for Tropical Disease, Hanoi, Vietnam on 12 September 2019 (approval
number: 09/HDDD-NDTU), the Medical Ethics Committee of Kanazawa University (approval
number: 3123-1), and the institutional review board of the Research Institute for Microbial Diseases,
Osaka University on 22 March 2022 (approval number: 2020-6-3).
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.
Data Availability Statement: The newly obtained sequences were deposited in GenBank with
accession numbers OQ832560-OQ832594 and OQ832609-OQ832627. The viral sequences analyzed in
the present study were retrieved using GenBank and BV-BRC and are listed in Table S2.
Acknowledgments: We thank all of the patients who participated in this study. We also thank the staff
members of the National Hospital for Tropical Disease, Hanoi, Vietnam for their collaborative support
in this study. We thank Silmi Rahmani and Koichiro Seko for their help with sample organization
and sequencing. We also thank Kumi Yamamoto for her assistance and generous support.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or
in the decision to publish the results.
Microorganisms 2023, 11, 1267 15 of 16

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