Background
Dengue fever (DF) and its more severe forms, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), have emerged as major public health problems in tropical and subtropical areas [
1,
2]. Infection with dengue viruses (DENV), which are maintained in a human-mosquito transmission cycle involving primarily
Aedes aegypti and
Aedes albopictus, can result in various clinical manifestations ranging from asymptomatic to DF, DHF, DSS and death [
3]. The occurrences of dengue epidemics in the past 30 years have been characterized by the rising incidence rates of infection and continuous expansion in geographic distribution of DHF epidemics [
4]. Importantly, the epidemics of DHF have become progressively larger in the last 20 years in many dengue endemic countries [
5]. The increasingly widespread distribution and the rising incidence of DF and DHF are related to increased distribution of
A. aegypti, global urbanization and rapid and frequent international travel.
Epidemiological analysis reveals that some DENV strains are associated with mild epidemics with low occurrences of DHF cases and inefficient virus transmission, whereas others are more likely to cause severe epidemics with high incidence of DHF/DSS and rapid virus transmission [
6,
7]. The large DHF epidemics in Indonesia in the 1970s and Sri Lanka after 1989 provided evidence supporting this phenomenon [
8,
9]. Dengue virus serotype 3 (DENV-3) re-appeared in Latin Americain 1994 after its absence for seventeen years. The virus was detected initially in Panama and soon dispersed throughout Central and South America during the following years [
10,
11]. This introduction coincided with an increased number of DHF cases in this region. Although the genotype originating in Southeast Asia has been postulated as the major cause of the increased virulence, the molecular marker associated with a difference in virulence among genotypes at the full-genomic level is still largely unknown.
Dengue is caused by four antigenically related but genetically distinct viruses (DENV-1, -2, -3 and -4) belonging to the genus
Flavivirus, family
Flaviviridae [
12]. DENV is a single stranded, positive-sense RNA virus, approximately 10,700 nucleotides in length. The genome contains a single open reading frame (ORF) that encodes a polyprotein, which is co- and post-translationally processed to produce three structural proteins, including capsid (C), pre-membrane (prM) and envelope (E), and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) [
12,
13]. A considerable number of studies have revealed that each serotype of DENV is composed of phylogenetically distinct clusters that have been classified into "genotypes" or "subtypes," and each genotype is also composed of phylogenetically distinct "groups" or "clades." A previous study has classified DENV-3 strains into four genotypes based on limited numbers of nucleic acid sequences from the prM and E protein genes [
6]; DENV-3 strains have also been re-classified into five genotypes [
14]. Growing evidence suggests the existence of DENV strains with different epidemic potentials. This evidence is supported by the following observations: (1) the differences in fitness among various genotypes of DENV-2 reflect their different replication capabilities in human monocytes and dendritic cells [
15]; (2) around 1991, clade replacement among DENV-3 genotype II containing isolates from Thailand was associated with changing serotype prevalence and incidence of DHF epidemics [
16]; and (3) sudden changes in the genotype of DENV at a single locality have been observed that appeared to originate from the genetic bottleneck of a large viral population [
14,
17]. This sudden genotype replacement has been associated with more severe DHF epidemics in Indonesia and Sri Lanka [
9,
18]. However, most of these studies involved the E gene alone. This raises an important question: Is the introduction of different DENV genotypes in disparate geographical locations a result of sequence differences outside of the E gene altering their epidemic potential, or it is simply a stochastic event in viral evolution?
Dengue epidemics in Taiwan are usually initiated by imported index cases (King et al., 2000). The re-emergence of dengue outbreaks in Taiwan started when DENV-2 was re-introduced into the off-islet of Hsiao-Liu-Chiu in 1981. In 1987–1988, another large-scale DENV-1 outbreak occurred in Kaohsiung and Pingtung in southern Taiwan [
19]. Although DENV-3 was detected sporadically from imported index cases, no DENV-3-related epidemic occurred until 11 DHF cases were confirmed in Kaohsiung in 1994 and 23 DHF cases in Tainan in 1998 [
20]. Taiwan neighbors many Southeast Asian countries and more than 25,000 travelers visit these adjacent countries annually. The surveillance system implemented by the Center for Disease Control in Taiwan (Taiwan-CDC) routinely detects many imported dengue cases each year. Thus, Taiwan is an ideal place to study the evolution and dispersion of DENV that may have different epidemic potential, particularly in the 1994 and 1998 DHF epidemics in Taiwan that coincided with the DHF epidemics in Southeast Asian countries [
21]. Complete genomic sequencing of DENV-3 strains collected from different geographical locations and isolation years offers the opportunity to understand the genetic stasis and possible selection pressure sites in the DENV genome other than within the E gene.
Discussion and conclusion
Viral sequence comparisons among isolates from dengue epidemics of different disease severities may provide valuable information regarding the molecular basis of the epidemic potential of the virus. DENV-3 re-appeared in 1998 in Taiwan and caused the DF/DHF epidemic in Tainan City after its first introduction in 1994 [
20]. This stimulates a great interest in understanding the molecular relationship of DENV-3 isolates in Taiwan during inter-epidemic periods and in comparing them with the strains circulating globally to understand evolutionary trends and geographical expansions. Here, we confirmed that the dengue epidemics in Taiwan were strongly associated with the globally circulating DENV-3 due to constant introduction of viruses from Southeast Asia by Taiwanese travelers. Our data demonstrates the sequence diversity among the full-genomic sequences of DENV-3 and the positive selection pressures exerted in different lineages (i.e. genotypes) at sites in DENV-3 non-structural genes.
Since most Taiwan dengue epidemics were initiated by the introduction of virus from imported cases [
21], phylogenetic analysis provides essential information to understand the history and origin of all Taiwan DENV-3 isolates originating in other countries (Fig.
1). The high nucleotide sequence identity (> 99.8%) among the strains isolated in 1998 indicates that they were from a single origin and further spread to different townships, such as Pingtung (ID#98TW358). The only 1998 imported DENV-3 isolated from a traveler who had recently visited Indonesia was more closely associated with the genotype II isolates from Myanmar and older isolates from Thailand. This virus differed from the virus isolated during the 1998 Tainan outbreak, which might suggest that multiple genotypes of DENV-3 circulated in Indonesia. This observation is consistent with a previous study indicating that at least two subtypes of DENV-3 were present in Indonesia [
18]. The phylogenetic analysis also suggested that a single 1999 isolate (ID#99TW628) from the same location as the 1998 epidemic was grouped together with the genotype III Sri Lanka isolates. Additional DENV-3 isolates from the first DENV-3-caused DHF outbreak in Taiwan (1994–1995) were grouped into genotype I. All these results implicated that repeated introductions of different genotypes of DENV-3 into Taiwan since 1994 were important causes of dengue epidemics, and that DENV-3 was not endemic in Taiwan. This situation may be similar in the subtropical region of China. Our country initiated airport fever screening during the severe acute respiratory syndrome (SARS) outbreak in 2003–04, and it successfully identified 40 confirmed, imported dengue cases [
22]. Airport fever screening can thus quickly identify imported dengue cases, and may prevent a significant number of dengue outbreaks that would have been initiated by imported index cases. However, its cost-effectiveness in preventing any dengue epidemics in Taiwan will need to be evaluated in the future.
With different DENV-3 genotypes imported into Taiwan from Southeast Asia and other parts of the world, this virus collection provides an excellent opportunity to examine the sequence diversity of different genes of the full-length DENV-3 viral RNA genome for genotypes other than genotype IV. The highest p-distance of nucleotide diversity of the full-length genomes occurred for the NS2A gene (5.84% ± 0.54%), followed by the E gene (5.04% ± 0.32%). In contrast, the highest p-distance of amino acid diversity of the full-length genomes occurred for the capsid gene (3.13% ± 0.96%), followed by the NS2A gene (2.57% ± 0.62%). This observation is consistent with the previous analysis in DENV-1, DENV-3 and DENV-4 [
16,
34], although the precise cause of the increased rate of amino acid change in the NS2A gene is unknown. A similar observation could also be made while analyzing the full genomic sequences of West Nile virus (WNV) isolated from different animal species [
35]. The flavivirus NS2A, a protein important for viral replication and particle formation [
12], is cleaved by viral serine protease. A mutation at the basic P1 cleavage site residue in NS2A blocks this processing event and is lethal for virus production while still allowing RNA replication [
36,
37]. Furthermore, this basic residue in NS2A and an acidic residue in NS3 are important determinants for virus assembly and/or release [
38]. Although the relative high sequence diversity of the NS2A gene of DENV-3 may be due to the lesser structural constraint required for NS2A, it is possible that positive selection pressures may be exerted on this gene. Especially in light of recent studies, NS2A together with NS4B and NS4A were identified as dengue virus-encoded proteins that could antagonize the interferon (IFN) response during viral infection [
39,
40]. Our analysis didn't detect any selection pressure exerted on the NS2A gene probably due to the small sample size; future studies will be needed to focus the selection pressure analysis on non-structural proteins and DENV evolution.
Several evolutionally conserved amino acid changes are preserved, which are unique in different DENV-3 genotypes (Table
3). These substitutions resulted in changes of its polarity, hydrophobicity or charge. Especially notable was the change from L to S at position 178 of the NS1 region, which is an amino acid substitution unique to genotype II. This might be the result of positive selection within the lineage of genotype II but not other genotypes. All DENV-3 isolates from Thailand belong to genotype II, and interestingly, based on a previous publication [
16], strains of DENV-3 isolated prior to 1992 in Thailand may have been replaced by two new locally evolving strains. This could be a sign of a new genotype evolving in Thailand; however, most of the mutations or substitutions occurring were deleterious and a purifying selection of DENV-3 was suggested [
16]. It is very likely that the previous analysis focused on only the E protein gene. Determining the possibility of a positive natural selection site in the non-structural genes of the new Thailand lineage will require further study. A number of T- and B-cell epitopes are present on the non-structural proteins, especially the NS1 gene [
41‐
43]. Even though the biological significance of the L to S change at position 178 of the NS1 region is unclear, growing evidence supported by
in vitro and
in vivo studies suggest that there are certain evolutionary forces acting on the NS1 gene shaping the gene flow of the dengue viral population, which might differ during viral replication in mammalian and mosquito cells [
44,
45]. This is the first time that a positive selection pressure site was detected in a non-structural protein in DENV-3 and its importance together with its functional relevance to epidemic severity will need to be examined with a larger sample size.
The global distribution of different genotypes of DENV-3 indicates that they originated in Southeast Asia; these genotypes demonstrated higher epidemic potential with regards to severe DHF epidemics in Sri Lanka, Central and South America [
46,
47]. Genotype III, once its transmission cycle was established locally, soon resulted in DHF epidemics regardless of an increase in virus transmission or a change in circulating serotypes [
7,
14], supporting the hypothesis that virus strain is an important risk factor for DHF [
48,
49]. Two sub-lineages (isolated before and after 1989) existed within the DENV-3 genotype III strains from Sri Lanka, and the viruses isolated after 1989 were associated with the DHF epidemic [
7]. We found that the strain isolated in 1999 from a indigenous dengue patient (99TW628) that did not lead to a large-scale epidemic of DF or DHF was more closely related to the lineage of the DENV-3 genotype III Sri Lankan strain isolated before 1989. Similarly, in Indonesia two sub-lineages of DENV-3 were present (isolated before and after 1998), and a greater DHF epidemic, especially in adult cases, was caused by the DENV-3 strains isolated after 1998 [
50]. The DENV-3 strain isolated in Taiwan during the DHF outbreak in 1994 was actually more closely related to the old Indonesian strain of genotype I from 1976–78. While it is currently unknown how the different sub-lineages within each genotype are associated with different DHF epidemic potential, a recent publication suggested that changing serotype prevalence could lead to differential susceptibility to cross-reactive immune responses [
16]. Furthermore, Wearing et al suggested that both vector and short-termed host cross-immunity are two factors responsible for dengue epidemics [
51]. It would be necessary to strengthen comprehensive dengue virological surveillance, especially in those endemic and hyper-endemic areas/countries, to monitor the emergence of DENV strains with epidemic potential for better epidemic prevention and vaccine development.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
DYC and CCK designed and performed all the experiments and drafted this manuscript together. DYC participated in the sequence alignment and statistical analysis. JHH and YCW helped with collecting field human isolates and LJC helped with sequencing experiments, together. THL helped for the field mosquito collection and GJC formulated the idea for this study and also provided critical comments regarding this manuscript. All authors read and approved the final manuscript.