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. 2025 Jul 18;16:100712. doi: 10.1016/j.ijregi.2025.100712

Genomic analysis and molecular profiling of dengue viruses in Sabah, Malaysia during 2019-2020

Mya Myat Ngwe Tun 1,2,3,4,⁎, Jecelyn Leaslie John 5, Takeshi Nabeshima 2, Abdul Marsudi Manah 6, Yuki Takamatsu 2, Takeshi Urano 3, Kouichi Morita 1,2,3,4,#, Kamruddin Ahmed 5,7,8,#
PMCID: PMC12336003  PMID: 40791846

Highlights

  • •

    The cosmopolitan genotype of dengue virus (DENV) serotype 2 was the dominant strain.

  • •

    Genotype IV of DENV serotype 1 was introduced into Sabah, Malaysia.

  • •

    A re-emergence of a distinct lineage of genotype I of DENV serotype 3 was observed.

  • •

    Amino acid mutations at identical positions were detected across DENV isolates.

  • •

    The first phylogenetic and complete genome analysis of DENV in Sabah.

Keywords: Dengue virus, Complete genome, Phylogenetic tree, Sabah

Abstract

Objectives

Limited information is available on the distribution of dengue virus (DENV) serotypes and genotypes in Borneo, particularly, in Sabah, because most studies have focused on Peninsular Malaysia. This study aimed to investigate the serologic and molecular epidemiologic characteristics of DENV in patients from Sabah.

Methods

Serum samples were collected from febrile patients in Kota Kinabalu and Lahad Datu between 2019 and 2020 at the Kota Kinabalu Public Health Laboratory. We performed virus isolation, serological testing, viremia quantification, and complete genome sequencing.

Results

Of 188 serum samples, 89 tested positive for DENV by quantitative reverse transcription-polymerase chain reaction: 20 DENV-1, 46 DENV-2, 20 DENV-3, and 1 DENV-4. A total of 38 viruses were successfully isolated. The isolates belonged to DENV-1 (genotypes I and IV), DENV-2 (cosmopolitan genotype) and DENV-3 (genotypes I and III). The phylogenetic analysis revealed close relationships with strains from Malaysia, Indonesia, and the Philippines. DENV-2 was the predominant serotype, whereas the highest viremia levels were observed in patients infected with DENV-3.

Conclusions

To the best of our knowledge, this study provides the first detailed phylogenetic and complete genome analysis of DENV in Sabah. Continued molecular surveillance is essential to enhance our understanding of dengue transmission and support effective control strategies.

Introduction

Dengue (DEN) virus (DENV) infection is one of the most common mosquito-borne diseases in urban and semi-urban areas of tropical and subtropical countries worldwide. DENV is transmitted to humans through the bite of female mosquito vectors of Aedes aegypti and Aedes albopictus. Globalization, urbanization, travel, and trade have facilitated the spread of DENV, mainly in Asia, the Americas, and Africa [1]. Over 7.6 million DEN cases were reported to the World Health Organization (WHO) by April 30, 2024, including 3.4 million confirmed cases, over 16,000 severe cases, and over 3000 deaths [2]. The clinical presentations of DENV infections range from asymptomatic to a severe illness that may lead to death if not properly managed. The different degrees of severity were re-categorized in 2009 by the WHO into DEN without warning signs, DEN with warning signs, and severe DEN, based on clinical findings and laboratory tests. DEN is clinically diagnosed and validated by identifying anti-DENV immunoglobulin (Ig) G and/or IgM and the non-structural 1 (NS1) antigen, using serology, or by detecting viral RNA through reverse transcription-polymerase chain reaction (RT-PCR) and quantitative RT-PCR (RT-qPCR) [3].

DENV belongs to the genus Flavivirus of the family Flaviviridae. Its genome is a single-stranded, positive-sense RNA, sized approximately 11 kb, with a single open reading frame encoding a precursor polyprotein of structural (capsid, pre-membrane, and envelope) and non-structural (NS1, NS2A, NS2B, NS3, NS4, and NS5) protein [4]. DENV is categorized into four serotypes (DENV-1-4) based on genetic and antigenic characteristics, and their inter-serotype nucleotide variability is approximately 30% [5]. Each serotype is further divided into several genotypes, which are geographically specific. DENV-1 has genotypes I, II, III (sylvatic), IV, V, and VI; DENV-2 has genotypes Asian I, Asian II, Asian/American, American, cosmopolitan, and sylvatic; DENV-3 has genotypes I, II, III, IV, and V; and DENV-4 has genotypes I, IIA, IIB, III, and sylvatic [[6], [7], [8], [9]]. Structural proteins contribute to the pathogenic functions of host attachment, virulence, and replication [10]. Non-structural proteins regulate protein synthesis, viral replication, pathogenesis, and control of the host’s viral responses [11]. Complete genome sequencing has improved our understanding of the genomic diversity of DENV and the consequences of such genetic variation in functional terms. Increases in severity and transmission are associated with amino acid variations in NS proteins [12].

DEN is hyperendemic in Malaysia, where it represents a substantial economic and health burden. Over 600,000 DEN cases and 1200 associated fatalities were reported in Malaysia from 2015 to 2021 [13]. All four DEN serotypes (DENV 1-4) are prevalent in Malaysia, although the predominant serotypes each year may vary [14]. Malaysia is administratively divided into 13 states and three federal territories and physically separated into two regions by the South China Sea: Peninsular Malaysia and East Malaysia. The prevalence of DEN varies across each area and most DEN studies have been conducted in Peninsular Malaysia [15]. Sabah, a state in eastern Malaysia, located on the northern tip of Borneo, recorded lower DEN incidence rates than Peninsular Malaysia. Given the marked environmental changes in Sabah, DEN cases have increased in recent years [16]. However, there is limited knowledge of the DEN burden, serotype, genotype distribution, and genomic characterization of DENV in Sabah. Therefore, the present study aimed to conduct a serological and molecular characterization of DENV infection in Sabah during 2019-2020.

Materials and methods

Study areas and specimen collection

From May 2019 to November 2020, a total of 188 serum samples were collected 2-7 days after the onset of fever from patients residing in two cities in Sabah, Malaysia: Kota Kinabalu and Lahad Datu. Kota Kinabalu, the capital and largest city on the west coast of Sabah, and Lahad Datu, a smaller town on the east coast known for its natural attractions, experience a tropical climate characterized by high humidity, stable temperatures, and frequent rainfall throughout the year. These samples were obtained from the Kota Kinabalu Public Health Laboratory (Supplementary Figure. 1). The samples were centrifuged to separate the serum, which was stored at −80°C until use.

Serological tests

The NS1 antigen from the serum samples was detected using an severe DEN Bioline Dengue NS1 antigen rapid test (Standard Diagnostic Inc., Korea). To confirm acute DENV infection, in-house DENV IgM was performed following a reported procedure [17]. Optical density (OD) was read at 492 nm and a positive control or sample OD/negative control OD ratio ≥2 was considered positive. In-house DENV IgG indirect enzyme-linked immunosorbent assay [17] was used to determine primary and secondary infection and exhibited a high correlation with DEN hemagglutination inhibition, the gold standard recommended by the WHO [18]. A DEN IgG titer of ≥29,000 or a titer between 3000 and 29,000, accompanied by a positive result for DEN IgM, NS1, or polymerase chain reaction, indicated secondary DEN infection, whereas a titer <3000 was indicative of primary DEN [18].

Quantification of DENV in serum samples by RT-qPCR

To confirm the presence of DENV in the serum, viral RNA was extracted using a QIAmp Viral RNA Mini Kit (Qiagen, Hilden, Germany), following the manufacturer’s instructions. Then, RT-qPCR was performed on the RNA, using a TaqMan Fast Virus 1-Step Master Mix (Life Technologies, Carlsbad, CA, USA), along with the DENV primers reported in previous studies [19,20]. Ten-fold serial dilutions of standard RNA (102-108 genome copies) were used to quantify the viral genome levels. The detection limit for the viral genome was 100 copies, and the viral genome levels were expressed as log10 genome copies/ml.

Virus isolation, RNA extraction, and DENV serotyping by RT-PCR

Cultured Ae. albopictus mosquito clone cells (C6/36) were resuspended in an Eagle’s minimum essential medium, supplemented with 2% fetal bovine serum and 0.2 mM non-essential amino acid. The cells were seeded in cultured flat tubes (Thermo Fisher, Waltham, MA, USA), mixed with 10 µl of patient serum, and incubated at 28°C for 7 days. The infected culture fluids were then harvested and transferred to fresh C6/36 cell cultures, and the tubes were incubated for an additional 7 days to prepare the second passage of cells [20]. To confirm virus isolation, the second infected culture fluid passage was collected, and viral RNA (Qiagen, Hilden, Germany) was extracted and amplified using a Takara One-Step RT-PCR Kit (Takara Bio Inc., Shiga, Japan), using consensus and DENV serotype-specific primers [21].

Complete genome sequencing and phylogenetic analysis

For complete genome sequencing, next-generation sequencing (Ion Proton, Life Technologies, CA, USA) of the isolated DENV was performed, as previously reported [20]. Global sequences were obtained from the International Nucleotide Sequence Database Consortium and annotation was performed using SeqKit. Sequences were aligned using MAFFT v7.407 [22]. The maximum likelihood phylogenetic tree was created using PhyML v3.2.0 [23], with 1000-replication bootstrap values and a substitution model (jModelTest v2.1.10) [24]. All sequences were submitted to the National Center for Biotechnology Information, GenBank, with accession no. MW369303-MW369341, MW369428-MW369435.

Operational definitions

Based on the WHO criteria, laboratory-confirmed DENV infection was defined through positivity on at least one of the molecular methods (virus isolation confirmed by a conventional RT-PCR test and/or viral genome detection from serum using a RT-qPCR test) or serology tests (NS1 antigen detection by an immunochromatographic test kit or anti-DENV IgM antibody detection by an IgM enzyme-linked immunosorbent assay test).

Statistical analysis

Data were analyzed using Prism v10.3.1 (GraphPad, San Diego, CA, USA). Analysis of variance was used for comparisons among multiple groups, and Student’s t-test was applied for comparisons between two groups. This included analyses of the correlation between viremia levels in sera of patients and the four DENV serotypes, as well as between viremia levels in the sera of patients and type of infection (primary and secondary) A P <0.05 was considered statistically significant.

Results

Patients’ serological profiles

Of the 188 febrile cases, 120 (63.8%) and 68 (36.2%) were laboratory-confirmed DEN and non-DEN infections, respectively, based on serology and molecular methods. Of the 120 patients with confirmed DEN, 79 (65.8%) were positive for NS1, 38 (31.6%) for anti-DENV IgM, and 25 (20.8%) for NS1 and IgM (Figure 1a). Gender was known for 106 patients with confirmed DEN. The male to female ratio was 1.9:1, indicating a significantly (P = 0.0001) higher incidence in males (n = 69) than in females (n = 37). Of 120 DEN-confirmed cases, 45 (37.5%) and 75 (62.5%) were primary and secondary DEN infections, respectively. The respective age distributions of these primary and secondary infections were as follows: 13 and 7 for 1–10-year-olds, 14 and 12 for 11–20-year-olds, 9 and 6 for 21–30-year-olds, 7 and 7 for 31–40-year-olds, 1 and 3 for 41–50-year-olds, 1 and 3 for 51–60-year-olds, and 1 and 2 for 61–70-year-olds. Thus, primary DEN infections predominated in those aged 1-30 years, and secondary infections predominated in those aged >40 years (Figure 1b). Moreover, there were significantly more patients with confirmed DEN with a secondary infection than those with a primary infection (P = 0.006).

Figure 1.

Figure 1

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(a) Serologic profiles and (b) distribution of age and type of infection of febrile patients.

Ig, immunoglobulin; NS1, non-structural 1.

Molecular evidence of DENV in patients

Of the 188 patients with febrile illness, 89 (47%) were DENV-positive by RT-qPCR, including 20 DENV-1, 46 DENV-2, 20 DENV-3, and one DENV-4. Among DEN RT-qPCR–positives, seven had different serotype co-infections: six patients were co-infected with DENV-1 and DENV-2 and one patient with DENV-2 and DENV-3 (Table 1). DENV-2 was the predominant serotype. The viral copies in the sera of patients infected with DENV-3 were significantly higher (P <0.001) than in those with DENV-1, DENV-2, and DENV-4 (Figure 2a). No significant differences were observed between the type of infection (primary or secondary) and the four serotypes of DENV (Figure 2b).

Table 1.

Laboratory profiles of patients who tested positive for dengue virus isolate.

Sample ID Type of infection Non-structural 1 Serotype Genotype Quantitative reverse transcription–polymerase chain reaction (log10 genome copies/ml) in serum
DENV-1 DENV-2 DENV-3
Mono-infection 1 Primary - DENV-1 I ND
2 Primary + DENV-1 I 8.8
3 Primary + DENV-1 I 7.7
4 Primary + DENV-1 I ND
5 Secondary + DENV-1 I 8.1
6 Secondary + DENV-1 I 8
7 Secondary + DENV-1 I 7.5
8 Secondary + DENV-1 I ND
9 Primary + DENV-2 Cosmopolitan 6.8
10 Primary - DENV-2 Cosmopolitan 8.5
11 Primary + DENV-2 Cosmopolitan ND
12 Primary + DENV-2 Cosmopolitan 9.0
13 Primary + DENV-2 Cosmopolitan 8.8
14 Secondary + DENV-2 Cosmopolitan 7.5
15 Secondary + DENV-2 Cosmopolitan 4.8
16 Secondary - DENV-2 Cosmopolitan 5.9
17 Secondary + DENV-2 Cosmopolitan 9.1
18 Secondary + DENV-2 Cosmopolitan 8.9
19 Primary + DENV-3 I 11.0
20 Primary - DENV-3 I ND
21 Primary + DENV-3 I 8.4
22 Primary - DENV-3 I 8.3
23 Primary + DENV-3 I 8.8
24 Primary + DENV-3 III 6.7
25 Secondary - DENV-3 I 6.7
26 Secondary + DENV-3 I 9.7
27 Secondary + DENV-3 I 7.7
28 Secondary + DENV-3 I 5.6
29 Secondary + DENV-3 I 5.6
30 Secondary + DENV-3 I 6.9
31 Secondary + DENV-3 I 8.0
Co-infection 32 Primary + DENV-1 I 9.4 4.2
33 Primary + DENV-1 I 8.2 5.7
34 Primary + DENV-1 I 8.2 5.2
35 Primary + DENV-1 I 8.3 3.9
36 Secondary + DENV-1 I 8.1 5.0
37 Secondary + DENV-2 Cosmopolitan 8.0 6.1
38 Secondary + DENV-3 I 4.1 8.5
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DENV: dengue virus; DENV-1, -2, and -3: DENV serotype 1, 2, and 3; ND: not done due to shortage of serum.

Figure 2.

Figure 2

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Viremia levels in serum of patients according to (a) dengue serotype and (b) type of infection.

DENV-1, -2, and -3: dengue virus serotype 1, 2, and 3.

Virus isolation and phylogenetic analysis

A total of 38 patients underwent successful virus isolation: 13 had DENV-1, 11 had DENV-2, and 14 had DENV-3, based on conventional RT-PCR (Table 1). No DENV-4 was isolated. The DENV-1 isolates belonged to genotypes I and IV, with genotype I being predominant. Phylogenetic analyses of the nucleotide sequences of the E gene of DENV-1 indicated that they were genetically close to the strains from Malaysia, China, Indonesia, the Philippines, and Singapore (Figure 3a). The DENV-2 isolates were identified as the cosmopolitan genotype. These isolates were genetically close to the strains from Malaysia, China, Indonesia, Singapore, and Thailand (Figure 3b). Phylogenetic analysis indicated that the DENV-3 isolates clustered into two distinct lineages corresponding to genotypes I and III, with genotype I being the most prevalent. DENV-3 isolates were genetically close to the strains from Malaysia, China, Indonesia, and the Philippines (Figure 3c).

Figure 3.

Figure 3

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(a) Phylogenetic trees based on the full coding region of the envelope protein gene of DENV-1. The representative strains of genotype obtained from GenBank are named by country origin, strain name, year of isolation, and GenBank accession number. Strain names in red font represent the dengue isolates from this study, whereas those in blue font indicate Malaysian dengue strains from previous years. (b) Phylogenetic trees based on the full coding region of the envelope protein gene of DENV-2. The representative strains of genotype obtained from GenBank are named by country origin, strain name, year of isolation, and GenBank accession number. Strain names in red font represent the dengue isolates from this study, whereas those in blue font indicate Malaysian dengue strains from previous years. (c) Phylogenetic trees based on the full coding region of the envelope protein gene of DENV-3. The representative strains of genotype obtained from GenBank are named by country origin, strain name, year of isolation, and GenBank accession number. Strain names in red font represent the dengue isolates from this study, whereas those in blue font indicate Malaysian dengue strains from previous years.

DENV-1, -2, and -3: dengue virus serotype 1, 2, and 3.

Amino acid variants of DENV isolates

The 27 DENV isolates were sequenced using next-generation sequencing, and amino acid variability analysis was performed on the complete genomes of DENV isolates across all three serotypes included in this study. For DENV-1, genotype I and IV strains detected in Malaysia in 2014 and 2005, respectively (GenBank accession numbers KU666939 and JN697056), were used as reference strains. For DENV-2, a cosmopolitan genotype strain isolated in Malaysia in 2014 (GenBank accession number KX452040) was used as the reference. For DENV-3, genotype I and III strains detected in Malaysia in 2014 and 2010, respectively (GenBank accession numbers MH051732 and LT996910), were used as reference strains. A total of 20 non-synonymous (NSY) variants were detected in genotype I and 57 in genotype IV of DENV-1 (Figure 4a), 21 in the cosmopolitan genotype of DENV-2 (Figure 4b), and 82 in genotype I and 39 in genotype III of DENV-3 (Figure 4c). Moreover, NSY mutations were higher in NS proteins than in structural proteins. Genotype I and III of DENV-1, the cosmopolitan genotype of DENV-2, and genotype I and III of DENV-3 showed genetic similarities of 99.4%, 98.3%, 99.3%, 97.5%, and 98.8%, respectively, to the reference Malaysian DENV strains.

Figure 4.

Figure 4

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Amino acid variants of complete genome: (a) DENV-1, (b) DENV-2, and (c) DENV-3 isolates.

DENV-1, -2, and -3: dengue virus serotype 1, 2, and 3; NS1, non-structural 1.

Compared with the genotype I reference strain of DENV-1 from Malaysia (2014), all DENV-1 isolates in this study exhibited the same nine mutations at amino acid positions 241 (V36A) in the M region; 466 (L186S) and 762 (V482I) in the E region; 1068 (N293S) in the NS1 region; 1305 (L178S) and 1397 (L52S) in the NS2 region; and 3140 (G647E), 3377 (T884I), and 3387 (P894S) in the NS5 region (Supplementary Table 1). Compared with the cosmopolitan genotype of reference strain of DENV-2 from Malaysia (2014), all DENV-2 isolates in this study showed the same two mutations at the amino acid positions 1065 (N290D) in the NS1 region and 3140 (K649R) in the NS5 region (Supplementary Table 3). Compared with the genotype I reference strain of DENV-3 from Malaysia (2014), all DENV-3 isolates in this study exhibited the same 12 mutations at the following amino acid positions: 867 (I94T) and 896 (V123I) in the NS1 region; 1176 (K51R) in the NS2 region; 1658 (R185K) and 1855 (K382R) in the NS3 region; 2481 (I239V) in the NS4 region; and 2540 (I50T), 2912 (K422R), 3052 (R562Q), 3142 (T652V), and 3369 (Y879F) in the NS5 region (Supplementary Table 4). Amino acid NS mutations for each isolate of genotype IV of DENV-1 and genotype III of DENV-3, aligned with the Malaysian reference strains, are described in Supplementary Tables 2 and 5, respectively.

Discussion

Although the incidence of DEN in Sabah is lower than in other states in Peninsular Malaysia, the Ministry of Health’s National Dengue Statistics reported that the DEN incidence and related mortality have increased in the state [15,25]. This study reports the serological and molecular epidemiology of DENV-1, DENV-2, and DENV-3 serotypes among patients with febrile illness in Kota Kinabalu and Lahad Datu, Sabah during 2019-2020. This work addressed the limited information on serotype, genotype distribution, and genomic analysis of DENV in Sabah.

Malaysia is hyperendemic for DENV infection. In Malaysia, secondary DEN infections represent a higher risk than primary infections [26]. Regardless of localities, all age groups are affected by DEN, and DEN IgG seropositivity (91.6%) has been detected among Malaysian adults [27]. In this study, the secondary DENV infection rate was significantly higher than the primary infection rate, and adults were mostly affected by secondary infection. We attained 47% DENV genome detection and 20% DENV isolation among patients with an acute febrile illness. Although viral RNA was detected by RT-qPCR, not all qPCR-positive samples yielded successful virus isolation. This could be attributed to several factors, including low viral load, the absence of severe cases, and the timing of sample collection. Although samples were collected between days 2 and 7 after fever onset, virus isolation becomes more difficult after day 5. Additional factors such as sample storage conditions, the presence of preexisting antibodies, and the high proportion (62.5%) of secondary infections may have further reduced virus viability in culture because antibodies in secondary infections can neutralize the virus and inhibit replication in cell lines.

The serotype distribution of DENV varies from year to year in Sabah. All four serotypes of DENV were present in 2013-2018 and the predominant serotypes were DENV-4 in 2013, DENV-1 in 2014, DENV-2 in 2015, and DENV-3 in 2016-2018 [25]. RT-qPCR analysis revealed mono-infections with all four DENV serotypes, as well as co-infections with DENV-1 and DENV-2, and DENV-3 and DENV-4. We acknowledge that a key limitation of this study is the lack of accompanying clinical data from patients with febrile illness. As a result, we were unable to investigate the relationships between clinical disease severity and viral loads in patients with mono- and co-infection. Future studies integrating clinical, virological, and genomic data will be essential to better understand the clinical impact of specific DENV serotypes and genotypes and their potential roles in disease progression.

Our findings showed that genotypes I and IV of DENV-1 co-circulated during the 2019-2020 outbreak. Genotype IV of DENV-1 has been occasionally reported in Malaysia, although it is less common than genotype I, which remains the predominant genotype in Southeast Asia. Dhanol et al. [28] reported the detection of DENV-1 genotype IV in Johor Bahru, Malaysia in 2014. However, to date, there have been no reports of DENV-1 genotype IV detection in Sabah. Furthermore, the cosmopolitan genotype of DENV-2 that were present agrees with a previous study conducted in Sabah in 2017 [29].

Genotypes I and III of DENV-3 were reported in Malaysia from 2007 to 2013; however, a greater abundance of genotype III was observed in Malaysia [30]. Najri et al. [29] reported that genotype I, II, and III of DENV-3 were detected in Sabah during the 2017 outbreak and genotype II and III were dominant. In our study of the 2019-2020 outbreak in Sabah, genotype I of DENV-3 was more prevalent than genotype III. Notably, one isolate (Sabah_99, OP595766) formed a distinct lineage within genotype I. This genotype shift may be one of the factors contributing to the increased viral titers observed in patients infected with DENV-3 compared with those infected with DENV-2. Future studies should investigate the potential phenotypic differences between these two genotypes. The co-circulation of DENV-3 genotypes I and III contributed to large DEN outbreaks in several Asian countries, including Bangladesh and Sri Lanka in 2023, Myanmar in 2017, and Thailand in 2015-2016, and was associated with increased viral genetic diversity and complex immune responses, heightening the risk of severe disease in these regions [20,[31], [32], [33]]. Despite the co-circulation of DENV-3 genotypes I and III during the outbreak in our study, the absence of severe DEN cases may be attributed to population-level immunity, circulation of less virulent viral strains, protective host genetic factors, favorable environmental and epidemiologic conditions, and potential underreporting or misclassification of severe cases.

The DENV-1, DENV-2, and DENV-3 isolates from this study revealed high genetic similarity among Malaysian strains and neighboring countries. Human movements between Sabah and neighboring countries are increasing because of trade and travel and could contribute to the movement of DENV and the outbreaks. No new genotype was observed during the 2019-2020 outbreak; this might be due to the dominance of the existing genotype over other genotypes. In addition, the climate of Sabah may be another factor that has a selective advantage over these genotypes. Our analysis included all four serotypes and revealed co-circulation of different genotypes, reflecting the heterogeneity of DENV strains present in the area. Although the sample size is limited, the temporal and spatial distribution and the serotype/genotype diversity suggest that the isolates are broadly representative of the viral population during that time.

Based on complete genome analysis, amino acid variants were most frequently found in NS5, NS2, and E proteins in DENV-1; NS5 and NS1 in DENV-2; and NS5, NS2, and E proteins in DENV-3. The NS5 region encloses the polymerase region that is highly conserved between serotypes and plays a key role in maintaining viral fitness and replication capacity [34]. The E gene of DENV is involved in viral entry and immune recognition, and its mutations can alter infectivity and antigenicity and lead to vaccine escape [35]. The NS1 gene plays a role in immune modulation and diagnostics, and mutations in NS1 can affect immune evasion, viral replication, and contribute to vascular leakage [36]. The NS2 gene is involved in viral replication, assembly, and protease activity, and its mutations can affect viral replication, protein processing, and pathogenesis [37]. However, the amino acid mutations identified in our study were not included in the list of novel mutations previously reported in the literatures. Amino acid mutations found at the same position across all DENV isolates or most of DENV isolates in our study may indicate sites under strong selective pressure, reflecting viral adaptation to host immune responses or environmental factors. Such conserved mutations might confer advantages in viral fitness, replication efficiency, or immune evasion, leading to their persistence across different strains. Further studies will clarify their importance for unique biological phenotypes and determine the impact of amino acid mutations in DENV isolates.

Our study provides valuable new insights into the molecular epidemiology of DENV during the 2019-2020 outbreak in Sabah, Malaysia. We report the co-circulation of distinct genotypes of DENV-1 and DENV-3, along with the predominance of the cosmopolitan genotype of DENV-2. Notably, genotype IV of DENV-1 was identified in Sabah, possibly representing its first documented occurrence in the region. In addition, the re-emergence of different lineage of genotype I of DENV-3 was observed. Complete genome analysis revealed several amino acid mutations not previously identified in earlier Malaysian strains, emphasizing ongoing viral evolution. These findings highlight the critical need for sustained genomic surveillance to monitor genotype diversity and track viral evolution over time. Given that most DEN vaccines include only a single genotype per serotype, our results underscore the importance of monitoring genotype shifts that may affect vaccine effectiveness and immune escape. Furthermore, characterizing genomic changes is essential for maintaining the accuracy and sensitivity of molecular diagnostic tools.

To the best of our knowledge, this is the first report to present complete genome data of DENV-1, DENV-2, and DENV-3 isolates from patients with febrile illness in Sabah. Our findings advocate for routine and regionally inclusive genomic surveillance to deepen our understanding of DENV dynamics and inform public health strategies, vaccine development, and diagnostic preparedness.

Funding

This work was supported by the Joint Research of Nekken grant (2020) and the Japan Agency of Medical Research and Development, under grant numbers JP253fa627004, 24jm0210114h0001, JP24fk0108656, 24wm0125011, and JP24wm0125006; Japanese Society of Promotion and Science (JSPS) KAKENHI, under grant numbers 24K10246 and 24K02288; and SDGs Research Project of Shimane University. The identified viruses will be registered in Nagasaki University through the National BioResource Project (Human Pathogenic Viruses) of MEXT, Japan.

Ethical approval

The study protocol was registered under the National Medical Research Registry and approved by the Medical Ethics and Research Committee, Ministry of Health Malaysia (NMRR-18-2869-41360). This work was also approved by the Institutional Ethical Review Committee of the Institute of Tropical Medicine, Nagasaki University, Japan (191003223).

Author contributions

Mya Myat Ngwe Tun: conceptualization, investigation, formal analysis, methodology, writing - original draft, review and editing; Jecelyn Leaslie John, Takeshi Nabeshima: investigation, data curation, methodology, software; Abdul Marsudi Manah, Yuki Takamatsu: writing - review and editing; Takeshi Urano, Kouichi Morita: funding acquisition, supervision, writing - review and editing; Kamruddin Ahmed: conceptualization, supervision, writing - review and editing.

Data availability

All data generated during the study are included in this manuscript. The National Center for Biotechnology Information accession numbers of the DEN virus isolates are provided in the manuscript; however, the genome sequences will be made publicly available after the manuscript is published.

Declarations of competing interest

The authors no have no competing interests to declare.

Footnotes

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.ijregi.2025.100712.

Appendix. Supplementary materials

mmc1.jpg (1.1MB, jpg)
mmc2.xlsx (33.1KB, xlsx)

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Associated Data

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

Supplementary Materials

mmc1.jpg (1.1MB, jpg)
mmc2.xlsx (33.1KB, xlsx)

Data Availability Statement

All data generated during the study are included in this manuscript. The National Center for Biotechnology Information accession numbers of the DEN virus isolates are provided in the manuscript; however, the genome sequences will be made publicly available after the manuscript is published.

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