The Virology Unit, Institute for Medical Research, serves as one of the national reference laboratories for dengue PCR serotyping in Malaysia. From 2024-2025, 22 cases of unknown serotype were identified among routine PCR test samples submitted for diagnostic confirmation. To meet reporting obligations and confirm these atypical findings, full-genome sequencing was initiated. Of these, eight samples yielded partial to complete genomes, revealed as sylvatic DENV2, while one sample was identified as a divergent DENV3. Low viral load in the remaining samples limited sequencing success, precluding full lineage assignment. Nonetheless, their strong resemblance in geographic location and sampling date to the confirmed sylvatic cases suggests a likely association. The detection of sylvatic DENV2 in multiple unrelated cases across different districts in Malaysia supports the possibility of low-level sylvatic transmission to humans, potentially contributing to the failure of commercial serotyping assays to detect these strains. These findings highlight the importance of genomic surveillance, especially for atypical or genetically distinct dengue strains.
Although human infection by sylvatic dengue viruses is uncommon, it has been reported sporadically. In Malaysia, an ancestral sylvatic DENV1 strain was previously isolated from a human case, sharing over 97% genomic similarity with a DENV1 strain from a sentinel monkey captured in 1972 [7] Globally, the first human case of sylvatic DENV2 was recorded in Nigeria in 1966 [15] followed by a similar case in Senegal in 1970 [16]. A 2020 outbreak in West Africa further suggests that sylvatic infections remain under-recognized due to diagnostic limitations [17]. A 2008 study described the isolation of a sylvatic dengue virus type 2 (DENV2) from a human patient in Malaysia. The complete genome sequence of this virus closely matched that of a sylvatic DENV2 strain isolated from a monkey in the same region in 1970 [8]. This finding marked the first isolation of a sylvatic dengue virus in Asia in over three decades.
Sylvatic DENV4 has been identified in several regions, notably in Malaysia and West Africa. It has been detected in Aedes niveus mosquitoes collected from the forest canopy, suggesting their role as vectors in the sylvatic cycle [18]. In terms of human infection, a novel DENV4 strain, DKE-121 was isolated from a hospitalized 37-year-old farmer in Malaysia and sequencing revealed that it is more related to sylvatic DENV4 [13].
In this study, phylogenetic analysis delineated two distinct sylvatic DENV2 clades: Clade I (African origin) and Clade II (Malaysian origin). The 1960s and 1970s Malaysian sylvatic strains are considered baseline references for the classical sylvatic DENV2 genotype, before major anthropogenic changes and inter-ecological virus mixing. The 2008–2009 isolates represent more recent spillover events but still pre-date the 2024 emergence and lack the mutations shared with urban strains. This consistent mutational pattern suggests strong intra-lineage conservation and likely reflects a localized and recent zoonotic emergence of this sylvatic DENV2 variant in human populations. This phylogeographic clustering further reinforces the distinct evolutionary trajectory and possible endemic expansion of Clade II within Malaysia.
Based on the amino acid variability analysis of sylvatic DENV2, three distinct categories of mutations were identified. The first category comprised mutations that were exclusively conserved across all Malaysian sylvatic strains, both historical and recent, highlighting lineage-specific signatures. The second category included mutations uniquely present in the recent Malaysian sylvatic strains isolated from human infections, distinguishing from historical strains and suggesting possible adaptation or diversification.
The third category consisted of mutations that were shared between the Malaysian 2024 sylvatic strains and urban DENV2 strains. The exclusive presence of the shared mutations in both recent sylvatic human-infecting isolates and urban DENV2 strains, but not in earlier sylvatic viruses, raises intriguing possibilities about the underlying evolutionary mechanisms. One hypothesis is that these shared mutations may have arisen through recombination events between co-circulating urban and sylvatic DENV2 viruses, particularly during co-infections in human hosts. Although recombination in dengue viruses is considered rare, it is not unprecedented, especially under conditions, where cross transmission between ecological cycles occurs. Alternatively, the presence of these mutations may reflect adaptive convergence, whereby sylvatic strains, upon entering and replicating within the human host environment, acquire or retain mutations that confer a selective advantage similar to those already established in urban strains. This could include enhanced replication efficiency, immune evasion, or transmission potential in human hosts or Aedes mosquitoes. Importantly, the absence of these mutations in historical sylvatic DENV2 sequences suggests that this pattern is not a remnant of ancestral traits, but rather a more recent and dynamic interaction. Whether driven by recombination, convergent evolution, or a combination of both, this finding supports the idea that ecological spillover events can lead to genetic exchange or parallel adaptation, potentially facilitating the emergence of sylvatic strains with enhanced capacity for sustained human transmission. Further studies, including full-genome recombination analysis and fitness assessments, are needed to clarify whether these shared mutations result from inter-lineage recombination or adaptive evolution under selective pressures in the human–mosquito cycle. Future work is also expected to incorporate Bayesian time-scaled phylogenetic methods to estimate divergence times between sylvatic and urban dengue strains, which may reveal evolutionary patterns and zoonotic emergence timelines.
The identification of a divergent DENV3 Isolate (UDS93/1) raises the possibility that this strain may represent a previously undetected sylvatic DENV3 lineage. Although no molecular evidence has definitively confirmed the existence of sylvatic DENV3, its potential has long been speculated in literature. Neutralizing antibodies to DENV3 were previously detected in the seroconverted sentinel monkeys in Peninsular Malaysia, suggesting past exposure via a sylvatic route [19]. While current data are insufficient to conclusively classify UDS93/1 as sylvatic in origin, its genetic divergence from known urban DENV3 strains, combined with historical serological evidence, underscores the need for continued genomic surveillance and targeted ecological studies to investigate the existence and public health significance of sylvatic DENV3 in Malaysia.
The predominance of NS1-positive yet IgM/IgG-negative results reflect early phase infections, where seroconversion had not yet occurred at the time of sampling. This serological pattern aligns with the observed clinical data, where many patients were sampled within the first 5 days of illness. The high proportion of severe cases (86.4%) raises questions regarding potential pathogenicity differences between sylvatic and urban strains. Sylvatic strains which were known to be adapted to non-human primates may cause more aggressive pathology in humans due to incomplete host adaptation. Such mismatch may provoke heightened immune activation, resulting in vascular leakage, organ involvement, and shock. The fatal case, involving an 80-year-old male with positive NS1 but negative IgM and IgG, suggests a primary infection. Immunosenescence and comorbidities in the elderly may have contributed to a blunted immune response, rapid progression, and poor outcome. Unfortunately, sequencing failed for this sample, and a repeat was not possible, preventing genomic correlation with disease severity. Hence, this fatal case, although unsequenced, was categorized as a presumptive sylvatic DENV2 infection based on epidemiological context.
Emergence of sylvatic dengue in human population requires diagnostic accuracy to detect and differentiate it from the urban dengue serotypes. Antigen and antibody assays, such as the detection of NS1 antigen, IgM and IgG are used to identify acute dengue infections. NS1 is detectable in the serum from the onset of symptoms and before the appearance of antibodies, making it a valuable marker for early diagnosis. Based on the current study data, it is apparent that the NS1 assays such as rapid diagnostic test kits can detect sylvatic dengue as NS1-positive; however, they do not differentiate between sylvatic and urban strains. Even though the differences in the nucleotide sequence of sylvatic dengue viral genes are quite distinct from the DENV1-4 urban serotypes, this does not affect the NS1 protein conformation and secretion in infected human. In fact, a study found that NS1 antigen was detected in the sylvatic DENV2 sample at high concentration [11].
Real-time RT-PCR is commonly employed to detect DENV RNA in patient serum or plasma samples, yet most commercial kits are not designed to identify sylvatic lineages specifically. The extensive genetic variability across structural and non-structural genes of sylvatic dengue complicates primer design. Follow-up sequencing and phylogenetic analysis remain necessary for confirmation, though this is time-consuming and resource-intensive. There is an urgent need for diagnostic tools tailored to sylvatic dengue, enabling timely detection and outbreak mitigation. The conserved 5′ and 3′ UTRs offer promising targets, as evidenced by incidental detection of sylvatic strains using a broad-range 3′UTR-targeting assay in this study. Similarly, Cecilia et al. [20] reported high conservation of the 3’UTR within each serotype. Notably, our analysis revealed that the NS2a region was also found to be highly conserved within DENV2 serotype with no evidence of sylvatic-associated signature mutations. Therefore, the 3′UTR and NS2a regions are reliable targets for pan-dengue detection assays, with the potential to capture both endemic and sylvatic dengue strains. In addition, the development of multiplex PCR panels or specific primers tailored for sylvatic lineages would enhance diagnostic sensitivity and specificity, particularly in areas, where sylvatic and endemic dengue viruses co-circulate.
Uncontrolled deforestation and rural urbanization globally pose risk of widening the interface between humans and sylvatic transmission cycle. Global deforestation, primarily in developing nations, causes 13 million hectares of land loss annually, accounting for 31% of the world’s total forest cover [21]. Enhanced surveillance in forest-edge communities is essential, particularly, where human–wildlife–vector interactions occur. The detection of sylvatic dengue virus in human cases also underscores the critical relevance of the One Health framework, which recognizes the interconnectedness of human, animal, and environmental health. Integrated vector and wildlife surveillance, particularly in areas of human encroachment into forested regions, is crucial for early detection and risk mitigation.
This study has limitations that should be acknowledged. First, sequencing was unsuccessful in 13 samples, which limited the number of complete genomes available for analysis particularly to perform recombination detection which are necessary to validate some preliminary findings. Second, the study did not include environmental or entomological surveillance data, which could have provided additional context regarding transmission dynamics and factor influencing sylvatic dengue virus circulation. Future studies incorporating broader genomic sampling and integrated ecological data are necessary to build a more comprehensive understanding of sylvatic dengue emergence.