Original article

Complete genome sequences and phylogenetic analysis of dengue virus in Southern Vietnam during 2014-2015

Thao Phuong Huynha,#https://orcid.org/0000-0002-8983-062X, Linh Tranb,c,#https://orcid.org/0000-0001-8667-082X, Quan Hoang Nguyena,#, Tam Chi Buia, Sherief Ghozyd,ehttps://orcid.org/0000-0001-5629-3023, Sara Morsyd,f, Thuan Minh Tieud,ghttps://orcid.org/0000-0003-4907-664X, Huy Tien Nguyenh,*https://orcid.org/0000-0002-9543-9440, Huong Thi Que Vua,*
Author Information & Copyright
aDepartment of Microbiology and Immunology, Pasteur Institute, Ho Chi Minh City, Vietnam
bInstitute of Fundamental and Applied Sciences, Duy Tan University, Ho Chi Minh City, Vietnam
cFaculty of Natural Sciences, Duy Tan University, Da Nang City, Vietnam
dOnline Research Club (http://www.onlineresearchclub.org), Nagasaki, Japan
eNeurosurgery Department, El Sheikh Zayed Specialized Hospital, Giza, Egypt
fMedical Biochemistry Department, Faculty of Medicine, Tanta University, Tanta, Egypt
gFaculty of Health Sciences, McMaster University, Hamilton, ON, Canada
hSchool of Tropical Medicine and Global Health, Nagasaki University, Nagasaki, Japan

# Phuong Huynh, Linh Tran and Quan Hoang Nguyen contributed equally to this work.

*Address correspondence: Huy Tien Nguyen at the School of Tropical Medicine and Global Health, Nagasaki University, Nagasaki 852-8523, Japan; E-mail: tienhuy@nagasaki-u.ac.jp

© Copyright 2022 MedPharmRes. This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Received: Jun 30, 2021; Revised: Dec 25, 2021; Accepted: Jan 12, 2022

Published Online: Oct 30, 2022



Dengue is an infectious disease that causes a worldwide health and economic burden despite the efforts to eradicate the disease. From 2013 to 2015, dengue epidemic significantly increased from 33,626 to 50,205 cases in Vietnam. This study aims to determine the genotype variations of dengue virus (DENV) circulating in Southern Vietnam during 2014-2015.


C6/36 cells were infected with twenty-four strains of dengue virus isolated in 2014-2015 and kept frozen. The complete nucleotide sequence of dengue virus genomes was obtained by polymerase chain reaction (PCR). The genome was sequenced in the MiSeq system and analyzed by the basic local alignment search tool (BLAST) program. Data from GeneBank was used to create the phylogenetic trees.


Among the 17 analyzed strains from 8 southern provinces, four (23.53%) were DENV-1, three (17.65%) were DENV-2, five (29.41%) were DENV-3, and five (29.41%) DENV-4 were isolated. Four DENV-1 isolates belong to Asia genotype. Three DENV-2 strains were concentrated in a subgroup of Asian 1 genotype. Five DENV-3 isolates were identified as belonged to Asian 2 genotype and five DENV-4 isolates were found as belong to Asia 1 genotype. There were no amino acid mutations and the transition capacity between the nucleotide among four types of DENV serotypes suggested that the probability of conversion from C to T was the highest conversion rate.


These DENV isolates were genetically close to other previous strains isolated from Vietnam and its neighboring countries, including Thailand, China, Cambodia, and Singapore, Brazil, Sri Lanka due to dynamic transmission.

Keywords: Dengue outbreak; Southern Vietnam; genome sequence; phylogenetic tree; diversity


Dengue virus (DENV) is a single-stranded flaviviridae RNA virus and responsible for causing the mosquito-borne infectious dengue disease mostly in tropical areas [1]. The disease manifestations range from mild dengue fever (DF) to the life-threatening dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). Naturally, the virus is circulating in four major serotypes (DENV-1–4) and showing about 65–70 % sequence homology with each other [2]. These serotypes show the highest mutation rates among other flaviviruses [3, 4]. This has led to the formation of different lineages and genotypes within each serotype [5]. Accordingly, DENV-1 comprises of five genotypes (I–V); DENV-2 has six genotypes (Southeast Asian/American, Asian I, Asian II, Cosmopolitan, American, and Sylvatic); DENV-3 has four genotypes (I–IV); and DENV-4 also consists of four genotypes (I, II, III, Sylvatic) [6]. Genetic changes can predominantly affect the disease burden by changing genotypes and subsequent changes in disease virility. An example of that is the changes of DHF rates in Sri Lanka when the DENV-3 (genotype III) group A was replaced with group B viruses [7] . Another example is when the American DENV-2 was replaced with the more severe Southeast Asian DENV-2 and the associated changes following this event [8].

Vietnam is an endemic country of dengue; particularly Southern Vietnam being a hyper-endemic area with nearly ten-fold more dengue than Central Vietnam every year [9, 10]. According to the summary report of the Pasteur Institute in Ho Chi Minh City, in 2013-2015, the number of dengue cases had tended to increase from 33,626 cases in 2013 to 50,205 cases in 2015. DENV-4 has been found in each area of Vietnam (Southern, Northern, Central) with over 50 strains isolated and the data was available in GenBank [10] . In 2013, a large dengue outbreak occurred in central Vietnam resulting in 204,661 clinical cases, with DENV-4 being the most predominant serotype followed by DENV-1, DENV-3 and DENV-2, respectively [10]. Similarly, another outbreak was detected around the same period at Cat Ba Island with identified 192 cases [11]. The cases were attributed to DENV-3 serotype (genotype III), which presented a high homology with the DENV strains affecting the nearby city of Hanoi in the same year [11]. These finding suggested that the viruses were probably introduced to the islands from the mainland (likely Hanoi), hence, the prevention efforts should be focused mainly at the origin [11]. Additionally, DENV-3 (genotype III) was detected in cerebrospinal fluid of an accountant living in Hai Phong, which showed a close relationship with the aforementioned serotypes using phylogenetic analysis. Noteworthy to mention that DENV-3 (genotype III) was reported for the first time in Vietnam in 2013 while genotype II had been the one previously circulating in the country [12]. In 2011, DF epidemics were noted in Hanoi with 24 outbreak points hitting eight districts with serotypes DENV-1 and DENV-2 detected in human samples [13]. Back to 1998, a widespread epidemic occurred of DHF with the incidence rate of 438.98 cases per 100,000 population, representing a 152.4% increase compared to 1997 epidemic [9]. Although most prevalent serotype was DENV-3, other serotypes were noted as well but in lower percentages [9]. A recent study presented that DENV-1 strains in Northern Vietnam were most closely related to the dengue virus outbreak in Cambodia from 2006 to 2008 [14].

In this study, we aimed to identify a complete genome sequence analysis for all four types of DENV strains circulating in Southern Vietnam during 2014-2015. The identification of recent serotypes and genotypes of the strains will contribute in predicting the transmissibility and virulence of the virus responsible for epidemic.


2.1. Growth and identification of the dengue viruses

We employed Aedes albopictus (C6/36) cell line to grow viruses. C6/36 cells were cultured in 25cm2 flask (Nunc, Cat.No. 163371) with Dulbecco’s Modified Eagle Medium (Gibco, Cat.12100-061) supplemented with 10% Fetal Bovine Serum (FBS) (Gibco, Cat.10084-168) for growth at 28°C. Twenty-four strains of dengue viruses isolated in 2014-2015 and kept in −80°C storage were used to infect the cell lines. After the incubation period of 7 days at 28°C, infected cells were harvested and identified by direct fluorescent assay (DFA) as well as immunofluorescence assay (IFA) with the following monoclonal antibody: anti-Flavin conjugated fluorescein isothiocyanate (FITC), anti-dengue virus serotype 1-4 and anti-mouse IgG conjugated FITC (Sigma, F5262). The information about monoclonal antibody was provided by US CDC: DEN.1 Hawaii (D2-1F1-3); DEN.2 NGC (3H5-1-21); DEN.3 H87 (5D4-11-24); and DEN.4 H241 (1H10-6-7).

Study samples were collected by Centers for Disease Control (CDC) in 20 provinces in the Southern Region. The agency responsible for isolation and sequencing is the Pasteur Institute in Ho Chi Minh City, Vietnam.

2.2. Collecting whole-genome of dengue viruses

RNA from dengue viruses of which serotypes had been identified (1 to 4) was extracted using the QIAampR Viral RNA Mini Kit (Qiagen 52906). Full genome of the virus strains was identified using the Superscript III Reverse Transcriptase kit (Invitrogen, Cat. 18080-051) and the Platinum Taq DNA polymerase (Invitrogen, Cat. 10966-018). Brieflt, we used Superscript III RT and random primer to synthesize cDNA. Then, we performed PCR to collect full genome by Plantinium Taq DNA polymerase (Invitrogen, Cat. 10966-018) with primer in Supplementary Table S1. Specifically, for the Superscript III Reverse Transcriptase kit, the reaction mixture recipe was 1 μL random primer, 1 μL dNTP, 3 μL diethyl pyrocarbonate (DEPC)-treated water, 2 μL buffer 10X, 4 μL Mg2+, 2 μL DDT, 1 μL RI, 1 μL Enzyme Reverse Transcriptase and 5 μL RNA target with the following thermal cycle: 25°C for 10 minutes, followed by 50°C for 50 minutes, then 85°C for 5 minutes, and finally 4°C for 60 minutes. For Plantinium Taq DNA polymerase (Invitrogen, Cat. 10966-018), the reaction mixture recipe was 2.5 μL Buffer 10X, 1 μL Mg2+, 0.5 μL dNTP, 0.5 μL for each forward primer and reverse primer, 0.1 μL enzyme, 17.4 μL DEPC and 2.5 μL cDNA. DNA polymerase activation was followed by 35 amplification cycles of 30 seconds at 94°C, then 30 seconds at 55°C and 3 minutes at 72°C.

The genome virus was purified using the QIAquick ® PCR Purification (Qiagen 21480) and QIAquick ® Gel Extraction kit (Qiagen 28704). DNA fragments from 100bp to 10kb were purified from primers, nucleotides, polymerases, and salts by the QIAquick spin column under the effect of centrifugation.

2.3. Sequencing and analysis

DNA concentration was quantified by Qubit fluorometer. The fluorescent dyes were associated with specific target molecules. Calibration curve was used to generate the quantitation results. Later, the DNA concentration was diluted to 0.2 ng/μL, then the DNA was ligated and indexed by PCR reaction with the following reaction mixture: 25 μL DNA, 15 μLNPM, 5 μL Index 1 and 5 μL Index 2. The thermal cycle was 72°C for 3 minutes, followed by 95°C for 30 seconds, then 12 amplification cycles of 10 seconds at 95°C, 30 seconds at 55°C and 30 seconds at 72°C, then 72°C for 5 minutes and finally 4°C for 60 minutes.

After being attached with the index, the products were purified by AMPure XP beads kit (Beckman Coulter A63880). The genome of dengue viruses was sequenced using the Next-Generation Sequencing (NGS)-Illumina method in MiSeq system. Briefly, the DNA adhered to the adapter would be attached to the flowcell. Each small segment on the flowcell was synthesized to form a cluster to amplify the read signal. Each run provided a line of A, T, G, or C, which would be attached to the blocker. The signal from the attached unit would be received, analyzed and processed [15]. Finally, the sequencing results were analyzed by BLAST program [16]. The phylogenetic analyses and estimates of transition capacity between DENV nucleotides were conducted using MEGA7 version 7 (http://www.megasoftware.net/). The sequences were purified by CLC Genomics Workbench 10.1.1 software and aligned by the maximum likelihood estimation method with bootstrap analysis of 100 repetitions and reliability value greater than 70%. The analysis begins with a specialized tree created from the input data. The original tree branches were swapped until the tree with the highest likelihood score was obtained [17]. All reference sequences of different geographical regions were retrieved from GeneBank and used for genome analysis to create phylogenetic trees (Supplementary Table S2).


3.1. Dengue virus isolation

The results of virus isolation were presented in Table 1. Of 24 strains, we identified 20 positive strains, including 4 strains of DENV-1, 6 strains of DENV-2, 5 strains of DENV-3 and 5 strains of DENV-4. The results of IFA under fluorescence microscope were presented in Fig. 1. The positive samples after virus isolation were sequenced. The DENV genome was about 11kb in length. From 20 sequenced samples, after purification by CLC Genomics Workbench, 17 strains had appropriate quality for sequencing and further genotype analysis (Supplementary Table S2). Of these 17 DENV isolates, 4 (23.53%) were DENV-1, 3 (17.65%) were DENV-2, 5 (29.41%) were DENV-3, and 5 (29.41%) DENV-4 was identified in our study. Most of them distributed across the Southern Vietnam regions including Dong Thap, Ba Ria-Vung Tau, Binh Duong, Soc Trang, Ben Tre, Tay Ninh, Ho Chi Minh City, Dong Nai (Table 1).

Table 1. Results of virus isolation
ID Strains Year Region DFA IFA Serotype
1 ÐT-HT-281/14 D1(1F1) 2014 Dong Thap (-)
2 VT-HT-00518/14 D1(1F1) 2014 Ba Ria-Vung Tau 3+ 3+ DENV-1
3 BD-DNT-5527/14 D1(1F1) 2014 Binh Duong 3+ 3+ DENV-1
4 ST-HT-1191/15 D1(1F1) 2015 Soc Trang 2+ 3+ DENV-1
5 BT-HT-8442/15 D1(1F1) 2015 Ben Tre (-)
6 TN-HT-9062/15 D1(1F1) 2015 Tay Ninh 2+ 2+ DENV-1
7 VT-HT-4573/14 D2 2014 Ba Ria-Vung Tau 3+ 3+ DENV-2
8 VT-HT-4577/14 D2 2014 Ba Ria-Vung Tau 3+ 3+ DENV-2
9 BD-HT-4598/14 D2 2014 Binh Duong 3+ 3+ DENV-2
10 HCM-M-1226/15 D2 2015 Ho Chi Minh 4+ 3+ DENV-2
11 ÐN-HT-4581/15 D2 2015 Dong Nai 3+ 3+ DENV-2
12 ÐN-HT-6763/15 D2 2015 Dong Nai 3+ 3+ DENV-2
13 BD-HT-258/14 D3 2014 Binh Duong 3+ 3+ DENV-3
14 TN-HT-6073/14 D3 2014 Tay Ninh (-)
15 VT-HT-6426/14 D3 2014 Ba Ria-Vung Tau 4+ 3+ DENV-3
16 ÐN-HT-1070/15 D3 2015 Dong Nai 2+ 2+ DENV-3
17 BD-HT-5786/15 D3 2015 Binh Duong (+) (+) DENV-3
18 ÐT-HT-6970/15 D3 2015 Dong Thap 2+ 2+ DENV-3
19 BD-HT-3268/14 D4 2014 Binh Duong 4+ 3+ DENV-4
20 VT-HT-3837/14 D4 2014 Ba Ria-Vung Tau 2+ 2+ DENV-4
21 ÐN-HT-7366/14 D4 2014 Dong Nai (-)
22 LA-HT-1128/15 D4 2015 Long An 3+ 3+ DENV-4
23 BD-HT-5297/15 D4 2015 Binh Duong (+) (+) DENV-4
24 BD-HT-7053/15 D4 2015 Binh Duong 2+ 3+ DENV-4
Download Excel Table
Figure 1. Negative (a) and positive (b) results of immunofluorescence assay (IFA) under fluorescence microscope observation. IFA is an indirect immunofluorescence reaction for determination DENVtype. The positivity level is assessed based on the percentage of fluorescence cells
Download Original Figure
3.2. Nucleotide sequence and phylogenetic analysis

Phylogenetic tree revealed that DENV-1 strains were divided into three main genotypes according to the geographic area, including Asia, South Pacific and America/Africa, in which four genome sequences of DENV-1 isolates belong to small subgroup of Asia genotype and genetically close to previous strains from Vietnam, Thailand, China and Sri Lanka (Fig. 2). For DENV-2, the phylogenetic tree showed three DENV-2 isolates were belong to Asian 1 group and genetically closest to the Thailand strain (Fig. 3).

Figure 2. Phylogenetic tree of genome DENV-1 sequences. Each geographical strain was abbreviated by its accession number followed by country, year of isolation and their region. Four DENV-1 isolates from our study were labeled as ▲
Download Original Figure
Figure 3. Phylogenetic tree of genome DENV-2 sequences. Each geographical strain was abbreviated by its accession number followed by country and year of isolation. Three DENV-2 isolates from our study were shown as •
Download Original Figure

Regarding DENV-3, the phylogenetic analysis reported that five strains isolated from our study were identified to group of Asian 2 genotype and genetically close to previous strains from Vietnam, Cambodia, Thailand and Singapore (Fig. 4). For DENV-4, the phylogenetic analysis demonstrated that five DENV-4 strains isolated from our study were identified as Asia 1 genotype. DENV-4 isolates were genetically close to other previous isolates from Thailand and Brazil (Fig. 5).

Figure 4. Phylogenetic tree of genome DENV-3 sequences. Each geographical strain was abbreviated by its accession number followed by country, year of isolation and their region. The genotypes were denoted as Asian 1 (I), Asian 2 (II), American (III), America/Asian (IV) and Cosmopolitan (V). Five DENV-3 isolates from our study were labeled as •
Download Original Figure
Figure 5. Phylogenetic tree of genome DENV-4 sequences. Each geographical strain was abbreviated by its accession number followed by country, year of isolation and their region. The genotypes were denoted as Asian 1 (I), Asian 2 (II), American (III), America/Asian (IV) and Cosmopolitan (V). Five DENV-4 isolates from our study were marked as ▲
Download Original Figure
3.3. The transition capacity among nucleotide

The transition capacity among nucleotides of four DENV was presented in Table 2. The result of analysis of DENV-1 showed that the highest ratio mutation was from C to T at 31.99% and the lowest ratio mutation was from G to C at 0.67%. Similarly, in DENV-2, the ratio mutation C to T was highest at 27.54% meanwhile from A to C was lowest at 1.61%. In DENV-3, the highest ratio mutation C to T was 33.87% whereas from G to C was 0.92 % and in DENV-4, the ratio mutation C to T was highest at 29.87%, while G to T was 0.5 %, respectively.

Table 2. Predicted transition ability among nucleotide between DENV-1, DENV-2, DENV-3 and DENV-4 sequences, respectively
A to T 1.68 1.65 0.97 1.88
A to C 1.46 1.61 0.92 1.04
A to G 11.82 13.01 11.10 14.08
T to A 2.49 2.59 1.45 2.65
T to C 30.38 26.88 32.12 28.16
T to G 0.96 1.99 1.16 0.6
C to A 2.27 2.59 1.45 1.56
C to T 31.99 27.54 33.87 29.87
C to G 0.83 1.99 1.16 1.64
G to A 14.65 16.87 13.92 16.72
G to T 0.81 1.65 0.97 0.50
G to C 0.67 16.1 0.92 1.30
Download Excel Table

Overall, the result of analysis revealed that the probability of conversion from C to T was the highest conversion rate among all four types of DENV-1, -2, -3, -4 serotypes. For comparison at the level of amino acids, the percentage difference in each pair of amino acid sequences between the four strains ranged from 0 to 1.03% (Supplementary Table S3).


In this study, we identified 17 complete DENV genome sequences, including 4 strains of DENV-1, 3 strains of DENV-2, 5 strains of DENV-3 and 5 strains of DENV-4 from patients with dengue during 2014-2015 in Southern Vietnam. Based on maximum likelihood method, the phylogenetic trees were constructed to detect any changes in the circulating strains of dengue in Southern Vietnam. Maximum likelihood method was considered as the most suitable fit for phylogenetic trees with large number of taxa and long sequence strings for viral phylogeny with its added bootstrap technique and high accuracy that we used to determine the branching patterns [18]. This information will help health authorities identify and take the necessary measure to prevent another epidemic.

DENV-1 was divided into the following genotypes: sylvatic/Malaysia, America/Africa, South Pacific, Asia, and Thailand [18]. Our phylogenetic analysis of four strains of DENV-1 revealed that the circulating strains belong mainly to Asian lineage and Thailand group. It was also related to both South Pacific and America/Africa. This demonstrates the diversity of the strains isolated from our cases. This was consisted with a study performed in Taiwan where they isolated DENV-1 from travelers returning from Vietnam. They observed this diversity in the strains of circulating genotypes in Vietnam [19]. Another study isolated dengue virus from 81 Vietnamese patients, in which, Rabaa et al. found that the circulating DENV-1 lineage in Southern Vietnam had come from Cambodia which contracted this lineage from Thailand [20]. This explained our tree results, which has shown genetic proximity to Thailand strains. Another study supported our finding that the DENV-1 strains were mainly distributed from Thailand and Indonesia [21]. Moreover, Mizuno et al. also proved that Vietnamese strain was the origin for strains circulating in China, Cambodia and Malaysia. Strains circulating in Japan and Korea were also found to be from South East Asia [22]. For its relation to Americas/African strain, a phylogenetic analysis revealed that the circulating strains in Caribbean and Americas was introduced from South Asia, mainly Thailand, which may explain the phylogenetic relationship between the strains [21].

Identification of the phylogenetic relations to other genotypes of DENV-1 will predict the incidence and severity of DENV-1 epidemic in Southern Vietnam [23]. It is well-known that American/African genotype has higher likelihood to cause DHF and more severe form of disease [18]. These strains also had a high potential to cause an epidemic [18, 23]. Similarly, DENV-1 also reached the dominant serotype in the outbreak of Myanmar in 2013, in which, the primary DENV infection remained at high level among the severe dengue cases [24].

In addition, the analysis revealed that three isolates of DENV-2 sequences in Vietnam were concentrated in a subgroup of the Asian genotype which along with the 1964 Thailand strain, are called Asian 1. A phylogenetic analysis of 273 strains of Asian lineage was divided into two groups; one comprised all strains from Thailand and China strains isolated during 1985–2001 and the other included Vietnam, Thailand and Cambodia from 2001 to 2008 [25]. In our analysis, our strains were related to older strains of the 1964 Thailand strain. It was demonstrated that the parent strain of modern strains in Vietnam is from Thailand [25]. It was also found that the strains in nearby regions are from Thailand. These findings were also supported by findings from a study in Central Vietnam during the outbreak of 2010-2012 [26]. Moreover, our results were consistent with another study that studied the evolution of DENV-2 in Vietnam [27]. They found that Asian I lineage had totally replaced the predominant Asian/American lineage since 2003 [27]. This was highly important since it was found that Asian strains were more related to development of sever forms of disease unlike other strains [23, 28]. It was able to have persistent high blood viremia in dengue patients. It was also found that genotypes of DENV-2 had high transmissibility, signifying its epidemics [18, 23]. A most recent study reported that DENV-2 isolated from the 2017 dengue outbreak in Northern Vietnam originated from India in 2006 [29].

Other two serotypes extracted from our patients were DENV-3 and DENV-4. DENV-3 isolates from our study were found as genetically close to previous strains from Vietnam, Cambodia, Thailand and Singapore. This finding was in agreement with results from Podder et al. [30] and Shu et al. [31] on the E gene region. Phylogenetic analysis of DENV-3 using E gene revealed four subtypes based on geographical distribution including Americas, Indian subcontinent, Thailand and Southeast Asia/South Pacific [18]. This classification was useful to identify the virulence and epidemic potential for the circulating serotype across the years in each country. The year of 1990 was considered as the cut-off year for the circulating DENV-3 in America, before 1990, only American genotype was the one circulating with low incidence of new cases and only DF was diagnosed [32, 33]. Unlike after 1990, Southeast Asian serotypes were associated with high epidemic and more diagnosis of DHF; Indian type was also found after 1990 in Americas and Asia and was correlated to severity of the disease [34, 35].

In case of DENV-4, the rate of incidence of epidemic from this serotype was less predominant than other serotypes imposing an obstacle for its classification into well-established genotypes [18]. Besides, our DENV-4 isolates were proved to genetically close to other previous isolates from Thailand and Brazil. This is consistent with the hypothesis that the presence of the Americas is due to the spread of these strains from Asia to America [36]. After the discovery of full sequence of E gene, only three genotypes were reported with only one type found to be circulating along many continents. Still, more efforts are needed to classify this serotype [36, 37].

The result of analysis revealed that the probability of conversion from C to T was the highest conversion rate among all four types of DENV-1, -2, -3, -4 serotypes. Based on the wobble theory [38], the transition from C to T was more likely to cause significant mutations on the protein surface such as Ser transfer to Phe and Leu, or Leu to Pro. These findings could play a key role in the genetic evolution. In addition, the percentage difference in each pair of amino acid sequences between the four strains ranged from 0 to 1.03% (Supplementary Table S3). However, the degree of amino acid sequence difference is always lower than that at the nucleotide level, indicating that most mutations occur in the third-base of the coding codon. There is almost no change at the amino acid level, so this result shows that most of these isolated viruses are in the stage of accumulation of mutations.


Apparent limitations of the present study include that we analyzed a relatively small numbers of specimens. In addition, there was a small number of isolates in each serotype, which might not give a complete representation of the circulating DENV in Southern Vietnam.


In conclusion, our study describes, for the first time, the genetic diversity of all four serotypes of DENV in Southern Vietnam during 2014-2015. We demonstrated that DENV is hyperendemic in Southern Vietnam, rendering co-infection between serotypes and genotypes possible. We observed the co-circulation of multiple genotypes within DENV-1 to DENV-4; four DENV-1 isolates belong to Asia genotype, three DENV-2 strains were concentrated in a subgroup of Asian 1 genotype, five DENV-3 isolates were identified as belonged to Asian 2 genotype and five DENV-4 isolates were found as belong to Asia 1 genotype. These findings highlighted the geographic distribution and dynamic transmission of DENV strains occurred in Southern Vietnam. The transition capacity between the nucleotide among all four types of DENV-1, 2, 3, 4 serotypes also suggested that the probability of conversion from C to T was the highest conversion rate, which implied genetic evolution of DENV strains.



dengue virus;


polymerase chain reaction;


basic local alignment search tool;


immunofluorescence assay;


dengue fever;


dengue hemorrhagic fever;


dengue shock syndrome;


Fetal Bovine Serum;


direct fluorescent assay;


fluorescein isothiocyanate;


Centers for Disease Control;


diethyl pyrocarbonate.


Ethical approval for this study was provided on September 2014 by the Institutional Review Board of the Ministry of Health, Vietnam (3711/QD-BYT). Informed consent was obtained from patients prior to the collection of samples (Supplementary Figure S1).

Supplementary Figure S1. Ethics approval
Download Original Figure


The authors received no financial support for the research, authorship, and/or publication of this article.


The authors declare that there is no conflict of interest.


Experiments were done by HPT, BCT and NHQ. Data analysis and its interpretation were done by LT, NHQ. All authors contributed to results interpretation, manuscript writing and give approval of the final version. All authors critically revised and approved the final manuscript.



Rigau-Perez JG, Clark GG, Gubler DJ, Reiter P, Sanders EJ, Vorndam AV. Dengue and dengue haemorrhagic fever. Lancet. 1998; 352(9132):971-7


Green S, Rothman A. Immunopathological mechanisms in dengue and dengue hemorrhagic fever. Current Opinion in Infectious Diseases. 2006; 19(5):429-36


Drake JW. Rates of spontaneous mutation among RNA viruses. Proceedings of the National Academy of Sciences of the United States of America. 1993; 90(9):4171-5


Holmes EC, Burch SS. The causes and consequences of genetic variation in dengue virus. Trends in microbiology. 2000; 8(2):74-7


Holmes EC, Twiddy SS. The origin, emergence and evolutionary genetics of dengue virus. Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases. 2003; 3(1):19-28


Weaver SC, Vasilakis N. Molecular evolution of dengue viruses: contributions of phylogenetics to understanding the history and epidemiology of the preeminent arboviral disease. Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases. 2009; 9(4):523-40


Messer WB, Gubler DJ, Harris E, Sivananthan K, de Silva AM. Emergence and global spread of a dengue serotype 3, subtype III virus. Emerging infectious diseases. 2003; 9(7):800-9


Rico-Hesse R, Harrison LM, Salas RA, Tovar D, Nisalak A, Ramos C, et al. Origins of dengue type 2 viruses associated with increased pathogenicity in the Americas. Virology. 1997; 230(2):244-51


Ha DQ, Tien NT, Huong VT, Loan HT, Thang CM. Dengue epidemic in southern Vietnam, 1998. Emerging infectious diseases. 2000; 6(4):422-5


Takamatsu Y, Nabeshima T, Nguyen TTT, Dang DT, Pham LHL, Pham HT, et al. A Dengue virus serotype 4-dominated outbreak in central Vietnam, 2013. 2015; p.:24-6


Le Viet T, Choisy M, Bryant JE, Vu Trong D, Pham Quang T, Horby P, et al. A dengue outbreak on a floating village at Cat Ba Island in Vietnam. BMC public health. 2015; 15:940


Phu Ly MH, Takamatsu Y, Nabeshima T, Pham Hoai LL, Pham Thi H, Dang Thi D, et al. Isolation of dengue serotype 3 virus from the cerebrospinal fluid of an encephalitis patient in Hai Phong, Vietnam in 2013. Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology. 2015; 70:93-6


Kim Lien PT, Duoc VT, Gavotte L, Cornillot E, Nga PT, Briant L, et al. Role of Aedes aegypti and Aedes albopictus during the 2011 dengue fever epidemics in Hanoi, Vietnam. Asian Pacific journal of tropical medicine. 2015; 8(7):543-8


Dang TT, Pham MH, Bui HV, Van Le D. Whole genome sequencing and genetic variations in several dengue virus type 1 strains from unusual dengue epidemic of 2017 in Vietnam. Virol J. 2020; 17(1):7


An introduction to Next-Generation Sequencing Technology [Internet]. 2017.


Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990; 215(3):403-10


Blume JD. Likelihood methods for measuring statistical evidence. Stat Med. 2002; 21(17):2563-99


Rico-Hesse R. Microevolution and virulence of dengue viruses. Advances in virus research. 2003; 59:315-41


Huang J-H, Su C-L, Yang C-F, Liao T-L, Hsu T-C, Chang S-F, et al. Molecular Characterization and Phylogenetic Analysis of Dengue Viruses Imported into Taiwan during 2008–2010. The American Journal of Tropical Medicine and Hygiene. 2012; 87(2):349-58


Rabaa MA, Simmons CP, Fox A, Le MQ, Nguyen TTT, Le HY, et al. Dengue Virus in Sub-tropical Northern and Central Viet Nam: Population Immunity and Climate Shape Patterns of Viral Invasion and Maintenance. PLOS Neglected Tropical Diseases. 2013; 7(12):e2581-e


Villabona-Arenas CJ, Zanotto PMdA. Worldwide Spread of Dengue Virus Type 1. PLoS ONE. 2013; 8(5):e62649-e


Mizuno Y, Kato Y, Kano S, Takasaki T. Imported malaria and dengue fever in returned travelers in Japan from 2005 to 2010. Travel Med Infect Dis. 2012; 10(2):86-91


Rico-Hesse R. Molecular evolution and distribution of dengue viruses type 1 and 2 in nature. Virology. 1990; 174(2):479-93


Ngwe Tun MM, Kyaw AK, Makki N, Muthugala R, Nabeshima T, Inoue S, et al. Characterization of the 2013 dengue epidemic in Myanmar with dengue virus 1 as the dominant serotype. Infection, Genetics and Evolution. 2016; 43:31-7


Waman VP, Kolekar P, Ramtirthkar MR, Kale MM, Kulkarni-Kale U. Analysis of genotype diversity and evolution of Dengue virus serotype 2 using complete genomes. PeerJ. 2016; 4:e2326-e


Tuan LV, Thi Tuyet Van N, Hoang Quan N, Tho Duoc P. Phylogeny of Dengue virus type 2 isolated in the Central Highlands, Vietnam. Revista de Biología Tropical. 2017; 65:819-26


Ty Hang VT, Holmes EC, Veasna D, Quy NT, Tinh Hien T, Quail M, et al. Emergence of the Asian 1 Genotype of Dengue Virus Serotype 2 in Viet Nam: In Vivo Fitness Advantage and Lineage Replacement in South-East Asia. PLoS Neglected Tropical Diseases. 2010; 4(7):e757-e


Watts DM, Porter KR, Putvatana P, Vasquez B, Calampa C, Hayes CG, et al. Failure of secondary infection with American genotype dengue 2 to cause dengue haemorrhagic fever. Lancet (London, England). 1999; 354(9188):1431-4


Dang TT, Pham MH, Bui HV, Le DV. First Full-Length Genome Sequence of Dengue Virus Serotype 2 Circulating in Vietnam in 2017. Infect Drug Resist. 2020; 13:4061-8


Podder G, Breiman RF, Azim T, Thu HM, Velathanthiri N, Mai le Q, et al. Origin of dengue type 3 viruses associated with the dengue outbreak in Dhaka, Bangladesh, in 2000 and 2001. Am J Trop Med Hyg. 2006; 74(2):263-5


Shu PY, Su CL, Liao TL, Yang CF, Chang SF, Lin CC, et al. Molecular characterization of dengue viruses imported into Taiwan during 2003-2007: geographic distribution and genotype shift. Am J Trop Med Hyg. 2009; 80(6):1039-46


Rodriguez-Roche R, Blanc H, Bordería AV, Díaz G, Henningsson R, Gonzalez D, et al. Increasing Clinical Severity during a Dengue Virus Type 3 Cuban Epidemic: Deep Sequencing of Evolving Viral Populations. Journal of Virology. 2016; 90(9):4320-33


Chungue E, Deubel V, Cassar O, Laille M, Martin PM. Molecular epidemiology of dengue 3 viruses and genetic relatedness among dengue 3 strains isolated from patients with mild or severe form of dengue fever in French Polynesia. The Journal of general virology. 1993; 74(Pt 12):2765-70


Balmaseda A, Sandoval E, Perez L, Gutierrez CM, Harris E. Application of molecular typing techniques in the 1998 dengue epidemic in Nicaragua. The American journal of tropical medicine and hygiene. 1999; 61(6):893-7


Harris E, Roberts TG, Smith L, Selle J, Kramer LD, Valle S, et al. Typing of dengue viruses in clinical specimens and mosquitoes by single-tube multiplex reverse transcriptase PCR. Journal of clinical microbiology. 1998; 36(9):2634-9


Lanciotti RS, Gubler DJ, Trent DW. Molecular evolution and phylogeny of dengue-4 viruses. The Journal of general virology. 1997; 78(Pt 9):2279-84


Chungue E, Cassar O, Drouet MT, Guzman MG, Laille M, Rosen L, et al. Molecular epidemiology of dengue-1 and dengue-4 viruses. The Journal of general virology. 1995; 76(Pt 7):1877-84


Varani G, McClain WH. The G x U wobble base pair. A fundamental building block of RNA structure crucial to RNA function in diverse biological systems. EMBO Rep. 2000; 1(1):18-23


Supplementary Table S1. The primers for RT-PCR. The primer sets are self-developed
No. Primer name Location Sequence (5’ – 3’) Length of products (in base pairs)
From To
5 D1,9F 3672 3690 ACAGGATGGGGATGGGAAC 1257
7 D1,12F 4801 4820 AGTGCAGGTGATTGCTGTTG 1659
9 D1,15F 6306 6325 GGCTGGATGCCAGAACATAC 1490
11 D1,18F 7691 7710 GAAGCCAAAGAGGGACTGAA 1490
13 D1,21F 9091 9110 TGGAAGGAGAAGGACTCCAC 1536
25 D2,13F 5372 5391 ACGAAGCCCATTTCACAGAC 1964
33 D2,17F 7243 7262 GGCATCATGAAAAACCCAAC 2011
41 D2,21F 9166 9183 GATGACACCGCAGGATGG 1304
25 D3,13F 4958 4978 CTGTATGGCAATGGAGTGGTT 1985
33 D3,17F 6787 6807 GCATACTTACATTGGCTGCAA 1760
41 D3,21F 8371 8390 AACACCCAACATGGATGTCA 1541
9 D4,5F 2005 2024 CTTTTGCTGAGaATACCAAC 1816
25 D4,13F 5461 5480 TCATGACTGCAACCCCTCCT 2008
33 D4,17F 7231 7250 CTGCTGGGATCATGAAGAAC 1768
41 D4,21F 8815 8834 GATGGACATCAGCCAGTGAA 1422
Download Excel Table
Supplementary Table S2. List of all strains used for genotype analysis
No. Genbank No. Strains Year Geographic origin Genotype Label
1 AB074760.1 Mochizuki 2001 Japan Asia JA01
2 JN054255.1 DV1_SL_2010b 2010 Sri Lanka Asia SL10
3 KC759167.1 DF1203 2012 China Asia CH12
4 KF887994.1 DENV-1/8/Thailand/01/2013 2013 Thailand Asia TH13
5 KF955446.1 DENV-1/VN/BID-V3909/2008 2008 Vietnam Asia VN08
6 KJ649286.1 DENV-1-Jeddah 2011 Saudi Arabia Asia SA11
7 AB074761.1 A88 2001 Japan South Pacific JA01A
8 AB189121.1 98901530 DFDV-1 1998 Indonesia South Pacific ID98
9 JQ915074.1 PF08/180908-01 2008 French-Moorea South Pacific FM08
10 JQ915076.1 PF09/090609-76 2009 French-Tahiti South Pacific FT09
11 FJ810415.1 DENV-1/VE/BID-V2253/2005 2005 Venezuela America/Africa VE05
12 JN903579.1 RGCB419 2008 India America/Africa IN08
13 KF184975.1 Angola_2013 2013 Angola America/Africa AN13
14 KJ189367.1 DENV-1/PR/BID-V8188/2010 2010 Puerto Rico America/Africa PR10
15 KJ189368.1 DENV-1/MX/BID-V8195/2012 2012 Mexico America/Africa ME12
16 KJ189367.1 DENV-1/PR/BID-V8188/2010 2010 Puerto Rico America/Africa PR10
17 KF041233.1 D2/Pakistan/2011-3/2011 2011 Pakistan Cosmopolitan-I PA11
18 KJ010186.1 DENV-2/PK/2013 2013 Pakistan Cosmopolitan-I PA13
19 JQ955624.1 Od2112 2011 India Cosmopolitan-I IN11
20 KF479233.1 QHD13CAIQ 2013 China Cosmopolitan-I CH13
21 EU056810.1 1349 1983 Burkina.Faso Cosmopolitan-II BF83
22 GQ398258.1 DENV-2/ID/1016DN/1975 1975 Indonesia Cosmopolitan-II IN75
23 GQ398259.1 DENV-2/ID/1017DN/1976 1976 Indonesia Cosmopolitan-II IN76
24 GQ398260.1 DENV-2/ID/1070DN/1976 1976 Indonesia Cosmopolitan-II IN76A
25 AY858035.2 BA05i 2004 Indonesia Cosmopolitan-III IN04
26 EU179857.1 DS31-291005 2005 Brunei Cosmopolitan-III BR05
27 EU482672.1 DENV-2/VN/BID-V735/2006 2006 Vietnam Cosmopolitan-III VN06
28 FJ196853.1 GD01/03 2003 China Cosmopolitan-III CH03
29 JF327392.1 DENV-2/SG/D2Y98P-PP1/2009 2009 Singapore Cosmopolitan-III SI09
30 AF100467.1 IQT1797 1998 Peru American PE98
31 GQ868592.1 DENV-2/CO/BID-V3358/1986 1986 Columbia American CO86
32 HM582099.1 D2/FJ/UH21/1971 1971 Fiji American FI71
33 AF119661.1 China 04 1985 China Asian-American CH85
34 HQ999999.1 DENV-2/GU/FDA-GUA09/2009 2009 Guatemala Asian-American GU09
35 KC294221.1 DENV-2/PE/IQA 2080/2010 2010 Peru Asian-American PE10
36 KC294223.1 DENV-2/PE/NFI1159/2010 2010 Peru Asian-American PE10A
37 GQ868591.1 DENV-2/TH/BID-V3357/1964 1964 Thailand Asian-I TH64
38 AF204177.1 44 1989 China Asian-II CH89
39 AF204178.1 43 1987 China Asian-II CH87
40 HQ891024.1 DENV-2/TW/BID-V5056/2008 2008 Taiwan Asian-II TA08
41 JF730050.1 DENV-2/US/BID-V5412/2007 2007 US Asian-II US07
42 JN796245.1 DENV-3/US/BID-V5055/2008 2008 US II US08
43 EU482458.1 DENV-3/VN/BID-V1014/2006 2006 Vietnam II VN06
44 HM181935.1 DENV-3/KH/BID-V3829/2007 2007 Cambodia II CA07
45 GU131905.1 DENV-3/IPC/BID-V3808/2008 2008 Cambodia II CA08
46 AY923865.1 C0360/94 1994 Thailand II TH94
47 FJ744740.1 DENV-3/TH/BID-V2329/2001 2001 Thailand II TH01
48 AY766104.1 Singapore 8120/95 1995 Singapore II SI95
49 AY496879.2 PhMH-J1-97 1997 Philippines I PH97
50 DQ401690.1 InJ-16-82 2006 Indonesia I IN06
51 AY858042.2 KJ30i 2004 Indonesia I IN04
52 AB214882.1 D3/Hu/TL129NIID/2005 2005 East Timor I TL05
53 EF629370.1 BR DEN3 RO1-02 2007 Brazil V BR07
54 M93130.1 H87 1990 Philippines V PH90
55 AF317645.1 80-2 2001 China V CH01
56 EU081198.1 D3/SG/05K2933DK1/2005 2005 Singapore III SI05
57 GU363549.1 GZ1D3 2009 China III CH09
58 EU529702.1 DENV-3/US/BID-V1089/2003 2003 US III US03
59 EU529699.1 DENV-3/US/BID-V1080/2006 2006 US III US06
60 FJ182011.1 DENV-3/US/BID-V1621/2005 2005 US III US05
61 GQ868587.1 DENV-3/VE/BID-V2483/2007 2007 Venezuela III VE07
62 GQ868578.1 DENV-3/CO/BID-V3405/2007 2007 Colombia III CO07
63 FJ373304.1 DENV-3/VE/BID-V1590/2004 2004 Venezuela III VE04
64 GU131954.1 DENV-3/CO/BID-V3404/2006 2006 Colombia III CO06
65 AY618992.1 ThD4_0485_01 2001 Thailand I TH01
66 JQ513345.1 H781363 2011 Brazil I BR11
67 AY618991.1 ThD4_0087_77 1977 Thailand I TH77
68 AY776330.1 Taiwan-2K0713 2004 Taiwan II TA04
69 GQ398256.1 DENV-4/SG/06K2270DK1/2005 2005 Singapore II SI05
70 FJ882599.1 DENV-4/US/BID-V2446/1999 1999 Puerto Rico II PR99
71 JQ513341.1 H780120 2010 Brazil II BR10
72 JQ513340.1 H780090 2010 Brazil II BR10A
73 JQ513339.1 H779652 2011 Brazil II BR11
74 AY618989.1 ThD4_0017_97 1997 Thailand II TH97
75 AY618988.1 ThD4_0476_97 1997 Thailand III TH97A
76 AY618989.1 ThD4_0017_97 1997 Thailand III TH97B
77 AB189124.1 98900666 DSS DV-2 1998 Indonesia Outgroup IN98
78 KF955458.1 DENV-3/VN/BID-V1876/2007 2007 Vietnam Outgroup VN07
79 Our sample VT-HT-00518/14 D1(1F1) 2014 Vietnam 0518/14
80 Our sample BD-DNT-5527/14 D1(1F1) 2014 Vietnam 5527/14
81 Our sample ST-HT-1191/15 D1(1F1) 2015 Vietnam 1191/15
82 Our sample TN-HT-9062/15 D1(1F1) 2015 Vietnam 9062/15
83 Our sample VT-HT-4577/14 D2 2014 Vietnam 4577/14
84 Our sample BD-HT-4598/14 D2 2014 Vietnam 4598/14
85 Our sample ÐN-HT-4581/15 D2 2015 Vietnam 4581/15
86 Our sample BD-HT-258/14 D3 2014 Vietnam 0258/14
87 Our sample VT-HT-6426/14 D3 2014 Vietnam 6426/14
88 Our sample ÐN-HT-1070/15 D3 2015 Vietnam 1070/15
89 Our sample BD-HT-5786/15 D3 2015 Vietnam 5786/15
90 Our sample ÐT-HT-6970/15 D3 2015 Vietnam 6970/15
91 Our sample BD-HT-3268/14 D4 2014 Vietnam 3268/14
92 Our sample VT-HT-3837/14 D4 2014 Vietnam 3837/14
93 Our sample LA-HT-1128/15 D4 2015 Vietnam 1128/15
94 Our sample BD-HT-5297/15D4 2015 Vietnam 5297/15
95 Our sample BD-HT-7053/15 D4 2015 Vietnam 7053/15
Download Excel Table
Supplementary Table S3. Summary of results for percent difference in nucleotides and amino acids sequences between DENV strains.
A) DENV-1 strains.
Difference (%) Vietnamese strains Asia South Pacific America/Africa
Sequence Nucleotide Amino acid Nucleotide Amino acid Nucleotide Amino acid Nucleotide Amino acid
Mean 2.6 1.03 3.55 1.38 8.01 6.54 8.4 7.2
Minimum 0.5 0 0.7 0.4 7.4 6.8 7.6 6.9
Maximum 3.5 1.8 4.6 2.1 8.9 8.3 9.2 9.9
Download Excel Table
B) DENV-2 strains.
Vietnamese strains Asian 1 Asian 2 America Cosmopolitan
Asian 1 1.44-5.41
Asian 2 6.07-9.78 6.0-8.14
America 10.22-11.46 8.61-11.08 7.51-10.61
Cosmopolitan 8.0-11.43 6.16-10.58 7.05-11.27 9.48-12.38
Asian-America 7.57-9.27 7.6-8.39 7.13-8.77 7.37-10.63 6.15-10.4
Download Excel Table
C) DENV-3 strains.
Vietnamese strains I II III IV V
Minimum 0.27 7.40 1.58 7.79 10.73 6.02
Maximum 1.86 9.22 4.39 9.76 12.92 6.96
Mean 1.31 8.45 3.12 8.53 11.70 6.35
Download Excel Table
D) DENV-4 strains.
% difference Minimum Maximum
Vietnamese strains 0.54 0.542 2.331
Genotype I 5.15 3.106 5.808
Genotype II 9.538 8.633 10.36
Genotype III 11.06 10.71 11.67
Download Excel Table