DISCUSSION

In this study we confirmed that strains of DENV2 and DENV4 post-1990 grouped into clades which were significantly different from those prior to 1990, as DENV1 and DENV3 described here and previously [Reference Zhang10]. The origin and dispersion of Flavivirus in some countries has been described previously [Reference Ospina11, Reference Chen12]. In this study, we hypothesized that DENV1–3 clades could have been introduced from other countries or evolved in situ in the late 1970s or early 1980s (DENV1, 31·4 years old; DENV2, 26·0 years old; DENV3, 17·1 years old). However, DENV4 clade appeared 1713 years ago (DENV4, 1713 years old). Because these strains harboured specific mutations, they might have improved fitness in mosquitoes (e.g. Aedes aegypti) or humans, allowing them to survive better than other strains. In fact, these specific mutations were first confirmed in the envelope protein of these strains (Table 1), e.g. Asn⁸ → Ser⁸, Val³²⁴ → Ile³²⁴, Val³⁵¹ → Leu³⁵¹ in DENV1 genotype I Asn⁸³ → Lys⁸³ in DENV2 genotype Asian I, Ala⁴⁷⁹→ Val⁴⁷⁹ in DENV3 genotype II, Val⁹⁶ → Met⁹⁶, Lys²⁰³ → Thr²⁰³(Ala²⁰³), Phe⁴⁶¹ → Leu⁴⁶¹ in DENV4 genotype I. The above-mentioned mutations were located within the important structural domains of the outer E protein (Table 1). E was believed to be mediate receptor binding, membrane fusion, and it also induced protective immunity [Reference Whitehead13]. According to the crystal structure of E protein [Reference Rey14, Reference Modis15], it consisted of three domains (domains I, II, III), followed by the stem region (H1, H2) and the transmembrane domain (TM1, TM2). Domain I was the molecular hinge region involved in low-pH-triggered conformational changes [Reference Roehrig16], domain II was involved in dimerization and membrane fusion [Reference Rey14], domain III played a role in receptor binding [Reference Rey14], and transmembrane domains were involved in virion assembly [Reference Hsieh17]. As described previously [Reference Hang18, Reference Van Slyke19], lineage replacement and point mutations often improve viral fitness, which might be why strains in these clades could be repeatedly endemic in Thailand under purifying selection [Reference Zhang10, Reference Zhang20, Reference Klungthong21] and/or immune selection [Reference Twiddy22]. Due to limited availability of full-length viral genomes, only the envelope protein was analysed in this study. Therefore, some specific mutations might also exist in non-structural proteins, other structural proteins (e.g. C and prM) and non-coding regions of these clades because some virulent sites have already been identified in these regions [Reference Bordignon23–Reference Grant25]. Certainly, DENV mutants harbouring these mutations should be constructed to validate this deduction.

The epidemiological feature of DENVs in Thailand is considerably different from those in Singapore, a neighbouring country in South East Asia, where more genotypes were co-circulating in the last decade [Reference Lee26]. The high diversity of DENVs in Singapore might be due to increased globalization in Singapore, as this country was a regional and global commercial and travel centre and therefore able to frequently exchange and import DENV through human movement.

Moreover, substitution rates of DENV1, DENV2 and DENV3 clades presented here are consistent with those described previously [Reference Zhang20, Reference Sall27]. However, the DENV4 clade evolved much more slowly (6·4 × 10⁻⁵ substitutions/site per year) than the other three clades described here and the DENV4 strains isolated in Thailand prior to 2002 [Reference Klungthong21]. In fact, the substitution rate of strains prior to 1990 (18 strains outside clade 1991–2007) in DENV4 genotype I was 1·37 × 10⁻³ substitutions/site per year (95% HPD interval: 0·85 × 10⁻³ to 1·94 × 10⁻³), which was 21·4 times higher than for DENV4 clade 1991–2007. The lower substitution rate showed a lower replication cycle of virus, particularly tropism, and transmission modes [Reference Gray28, Reference Hanada29]. This might be one reason that DENV4 was the least frequently circulating serotype in Thailand since 1991 [Reference Drummond9].

As shown in Figure 1, the predominant genotypes of DENV1–4 showed a similar demographic pattern in genetic diversity: several ‘invasion’ phases followed by a ‘stationary’ phase which started from 2000. The ‘invasion’ phase was characterized by an increase in the genetic diversity of DENV. Markedly, apparent ‘invasion’ phases (1994 for DENV1, 1985 for DENV2, 1992 for DENV3, 1991 for DENV4) appeared just after the time of the first isolated strain in each DENV clade (1990 for DENV1, 1983 for DENV2, 1991 for DENV3, 1991 for DENV4), which suggested that new strains in DENV1–4 clades began to be responsible for the local dengue outbreak. Consistent with such a fact: DENV1 clade 1990–2007, DENV2 clade 1983–2006, DENV3 clade 1991–2003, and DENV4 clade 1991–2007 were predominant from 1994, 1985, 1992, and 1991, respectively. However, there were only four strains in DENV1 clade 1990–2007 but 15 strains outside this clade from 1990 to 1993 (Supplementary Fig. S1 a); there were only three strains in DENV2 clade 1983– 2006 but eight strains outside this clade from 1983 to 1984 (Supplementary Fig. S1 b); and there was one strain in DENV3 1991–2003 and one strain outside this clade in 1991 (Supplementary Fig. S1 c). Because strains in DENV1–4 clades had improved fitness in hosts (humans and mosquitoes), they survived better than other strains and were predominant so that the genetic diversity remained constant after 2000.

In summary, clade replacements in DENV1–4 took place in strains isolated in Thailand. The DENV strains in the new clades harbour specific mutations in the E protein and are responsible for local dengue epidemics post-1990.