During 1986–2007, dengue cases in Puerto Rico ranged from 2,000 to ≈16,000 per year (Figure 1, panel A), with major epidemics (>8,000 cases) reported in 1986, 1992, 1994, 1998, and 2007 (2–4,18,19). Despite major fluctuations in serotype circulation, DENV-2 circulated predominantly for 10 years (Figure 1, panel B), alternating with DENV-1 through 2 periods of resurgence during the 1990s and cocirculation of DENV-4 (Figure 1, panel B). DENV-2 declined markedly after the 1998 epidemic and the dissemination of DENV-3 concomitant to the disappearance of DENV-1 and -4. However, DENV-2 continued to cause a low number of cases during 1999–2003 and reemerged in 2004–2007 (Figure 1, panel B). Samples from every year of the 22-year study period (Figure 1, panel C) comprised our analysis.

The Bayesian Skyline analysis (Figure 1, panel D) of the autochthonous viral sequences (Figure 2; clades IB and II Figure A1) showed a gradual increase in the genetic diversity of DENV-2 during 1987–1991 that corresponds to a period of high transmission and dominance (Figure 1, panel B). This increase was followed by 9 years of high genetic diversity that coincided with a period of DENV-1 and DENV-4 cocirculation. The genetic diversity of DENV-2 declined sharply during 1999–2003, coinciding with a period of minimal DENV-2 transmission. Genetic diversity rebounded in 2005 to roughly pre-1999 levels as the virus reemerged.

The densely populated island of Puerto Rico (3,808,610 population; 3,508 square miles) is divided into 78 municipalities grouped in 8 regions. The 140 DENV-2 genomes from 37 municipalities represented all 8 regions and ranged from 2–20 isolates per year (Figure 1, panel C; Figure 2). We included 20 other sequences from Caribbean countries. The number of Puerto Rico sequences is proportional to the epidemic level or the relative proportion of DENV-2 identifications in the municipalities with highest DENV-2 reporting per year. The phylogeny of the 160 DENV-2 genomes showed 2 major clades (I and II) and a smaller clade (III) (Figure 2). Clade I contains 115 sequences and can be further subdivided in 2 subclades. Subclade IA (1998–2007) contains 13 Puerto Rico and 12 Caribbean sequences, including 1 from St. Thomas (SH 90 FJ898450) identified as the closest ancestor. Subclade IB (1994–2007) contains 90 sequences mostly of local origin, but the presence of 6 foreign and 1 local basal sequences confirms its foreign origin. Clade II (1986–1994) contains 39 local isolates. Basal to clades I and II is a St. John 1987 sequence (SJ 87 GQ868603), which suggests a possible origin. Clade III is formed by 4 local isolates during1987–1991. These genetically distinct isolates do not fit in clade I or II, but a separate analysis with publically available envelope gene sequences pointed to possible Caribbean origin (K.L. McElroy et al., unpub. data).

Four events merit recognition (Figure 2). First, a mixture of foreign and local strains at the base of subclades IA and IB provides evidence of multiple introductions. Eight Puerto Rico viruses associated with these foreign strains date from 1994 through 1999. These years also are associated with a distinct subgroup basal to subclade IB concomitant with the extinction of clade II in 1997. Second, subclade IB evolved mainly after the introduction of DENV-3 in 1998. Third, a period of limited circulation of DENV-2 reflected in low levels of genetic diversity (1999–2003) coincided with the expansion of DENV-3 and decline of DENV-1 and -4. Fourth, there was a resurgence of DENV-2 during 2004–2007.

Forty-nine amino acid differences mapped to the phylogeny were detected across the major internal branches of the tree. Twenty of these comprise major differences between clades I and II and between subclades IA and IB, as well as substitutions that arose during the continuous evolution of subclade IB (Figure 2). Only 1 aa substitution distinguished isolates in clades I/II from III: a hydrophilic glutamine to a hydrophobic leucine at position 131 in the E protein. Excluding PR79_1995_EU569708 as a possible foreign introduction, 18 aa differences distinguish isolates across clade I, 12 of which separate subclade IB from clade II and potentially could have been involved in the 1994–1997 lineage turnover (Figure 2). The remaining differences between isolates in subclades IB and II were present in nonstructural (NS) genes and are preponderantly conservative mutations, with the exception of position 31 in NS3, which was nonconservative. Among the additional changes, the only nonconservative mutation was a hydrophobic alanine to hydrophilic threonine at position 137 in NS4B that originated with PR40_1999 EU482730, and most changes were found in the NS genes.

Using Bayesian MCMC and d_N/d_S analyses, we estimated the mean substitution rates for the full genomes at 9 × 10^–4 to 1.1 × 10^–3 for all clades, consistent with previously published rates (20,21). The low d_N/d_S ratios (0.07–0.08) provide evidence of a low percentage of substitutions that have been fixed along independent lineages, possibly indicating purifying, negative selection.

BaTS analysis shows that lineages often correlated with the corresponding region of origin of the isolates. Seven of the 8 regions had >4 isolates in subclade IB or clade II. This association was significant for 6 regions (p<0.05) (Table). The most significant geographic correlation of lineages were found in the San Juan (1986–1990 and 1994–1996), Ponce (1987–1989), and Mayaguez (1989 and 1993) (Figure 3, panel A). In addition, isolates clustered geographically for San Juan (1997–1999 and 2001–2006), Caguas (1998–2001, 2004, and 2005), Ponce (1995–1997 and 2005), Mayaguez (1996–1998 and 2006), Aguadilla (1996–1998), and Arecibo (1994–1995, 2004, and 2006) (Figure 3, panel B ). Considering the DENV-2 historical data, we recognize that high-reporting municipalities usually are located in regions where we identified significant phylogenetic clustering (Figure A1). For example, in 1987, most DENV-2 cases originated from the Ponce and San Juan regions, where we identified lineages of clade I. For1994–1996, DENV-2 cases in San Juan, Ponce, Mayaguez, and Arecibo regions may reflect the coexistance of subclades IB and clade II.

We investigated other possible associations with the DENV-2 phylogeny, including age and DF/DHF status, but found none. Most DENV-2 infections were secondary (84.6% and 77% of DENV-2 infections in the CDC collection and this study, respectively). However, we found no relationship between phylogeny and incidence of primary or secondary infection in patients.

The year 1999 began a period of low circulation and low genetic diversity of the Caguas lineage of subclade IB (Figure 1, panel D; Figure 2; Figure 3, panel B) that lasted until 2003. During these 4 years, most DENV-2 cases originated from only 4 municipalities in eastern Puerto Rico (Figure 4, panel A); <20 additional DENV-2 cases were reported during that period in 12 other neighboring municipalities (Figure 4, panel A). Because phylogenetic lineages are geographically and temporally clustered, (Figure 2), we illustrated these associations on the map of Puerto Rico (Figure 4). This map shows that DENV-2 descendants from western Puerto Rico emerged in San Juan in 1997–1998 (Figure 4, panel B, top), then appeared and persisted within the refuge in 1999–2002 (Figure 4, panel C, middle) to then disseminate across the island in 2003–2005 (Figure 4, panel D). In the 4 municipalities with uninterrupted DENV-2 transmission, DENV-2 incidence increased 2 years after the islandwide increase (Figure 5). DENV-3 incidence within this DENV-2 refuge was minimal during the period of high DENV-2 incidence but peaked 2 years later concomitant with an increase across the rest of the island

Puerto Rico is a model for fine-scale studies on DENV evolution in the Americas. The long-term persistence of DENV-2 and its ability to reemerge after transient periods of low circulation is a remarkable aspect of the epidemiology of dengue in the region. The fact that 13% of DENV-2 isolates represent importations or close descendants from importations brings new insights to our understanding of DENV long-term circulation. Foreign viruses were identified in 8 years (1987, 1989, 1991, 1995, 1998, 1999, 2005, and 2007), of which only 1991 and 1998 had been previously sampled (14). Ten of the 18 introductions occurred during periods of high DENV-2 predominance: 1987–1991, 1995, and 2005–2007 (Figure 1, panel B; Figure 2). The other 8 introductions originated from the 1998 epidemic or shortly thereafter (1999). Therefore, DENV-2 seems to be introduced mainly during periods of favorable preponderance, not necessarily epidemic transmission of this serotype. Subclade IA viruses never established themselves, regardless of year of isolation or origin. These assessments showed a previously unknown feature of DENV-2 persistence: the endemic strain is recalcitrant to influences from frequent foreign introductions.

The relative inability of “foreign” DENV-2 to persist in the presence of the dominant subclade IB viruses is not well understood. The Puerto Rico strain might be highly adapted and thus have a fitness advantage, the frequently introduced strains might be simply underrepresented, or introduced strains may have disappeared through genetic drift. Isolate PR76_1995_EU569708, which lies basal to this subclade in the phylogeny (Figure 2), is more closely related to South American DENV-2 viruses than to other Puerto Rico viruses, and this lineage does not appear to have progressed, supporting the foreign origin of subclade IB. Our findings then show that subclade IB resulted from an introduced strain, as previously suggested by Bennett et al. (14), and successfully penetrated during a period of proportionally high incidence of foreign introductions. Interestingly, this clade replacement was completed in 1997, less than a year before the finding of DENV-3 and the concomitant decline of DENV-1, -2, and -4. The early portion of subclade IB is seen as a period of short-lived lineages ending in 1997, therefore, the rise and expansion of this subclade mainly occurs in coexistence with DENV-3, a different epidemiologic scenario from that of the now extinct clade II a decade earlier.

The dominance of conservative amino acid changes that segregated the viruses by clade hinders the assessment of phenotypic changes. Compensatory mutations might have conferred replicative advantages that could have influenced the displacement of clade II or the persistence of subclade IB in Puerto Rico; however this hypothesis has not been tested. Positive selection was not identified, contrasting with previous analyses (14,22–24). Others have not detected positive selection and attribute lineage extinctions or clade replacements to stochastic events rather than natural selection (25). More analysis to detect site-specific selection is needed to corroborate whether positive selection is not at play in these populations of viruses.

The period 1999–2003 represents historically low rates of DENV-2 circulation (Figures 1, 2, 4), and the epidemiologic and phylogenetic aspects of this transient retrieval had not been studied previously. We show that the genetic variability of DENV-2 decreased during these 4 years when the virus was transmitted in only a subset of municipalities. DENV-2 represented 29% of the cases in this area but only 5% island-wide. The reason this region became a refuge of DENV-2 for 4 years remains unclear, but the low incidence of DENV-2 in prior years compared with the rest of the island suggests susceptibility for infection in this population (Figure 5). Studies in Thailand showed serotype displacement affecting population diversity and lineage turnover (26). Short-term serotype cross-protection has been suggested to contribute to serotype displacements (27–29), implying that as DENV-3 infected a large susceptible population, cross-protective antibodies momentarily impeded transmission of other serotypes and dissemination of DENV-2 outside the eastern refuge. Our study confirms the utility of systematic sampling and genome sequencing in large-scale surveillance systems as ways to understand the dynamics of dengue transmission and endemicity.