Twenty Years of DENV-2 Activity in Brazil: Molecular Characterization and Phylogeny of Strains Isolated from 1990 to 2010

In Brazil, dengue has been a major public health problem since its introduction in the 1980s. Phylogenetic studies constitute a valuable tool to monitor the introduction and spread of viruses as well as to predict the potential epidemiological consequences of such events. Aiming to perform the molecular characterization and phylogenetic analysis of DENV-2 during twenty years of viral activity in the country, viral strains isolated from patients presenting different disease manifestations (n = 34), representing six states of the country, from 1990 to 2010, were sequenced. Partial genome sequencing (genes C/prM/M/E) was performed in 25 DENV-2 strains and full-length genome sequencing (coding region) was performed in 9 strains. The percentage of similarity among the DENV-2 strains in this study and reference strains available in Genbank identified two groups epidemiologically distinct: one represented by strains isolated from 1990 to 2003 and one from strains isolated from 2007 to 2010. No consistent differences were observed on the E gene from strains isolated from cases with different clinical manifestations analyzed, suggesting that if the disease severity has a genetic origin, it is not only due to the differences observed on the E gene. The results obtained by the DENV-2 full-length genome sequencing did not point out consistent differences related to a more severe disease either. The analysis based on the partial and/or complete genome sequencing has characterized the Brazilian DENV-2 strains as belonging to the Southeast Asian genotype, however a distinction of two Lineages within this genotype has been identified. It was established that strains circulating prior DENV-2 emergence (1990–2003) belong to Southeast Asian genotype, Lineage I and strains isolated after DENV-2 emergence in 2007 belong to Southeast Asian genotype, Lineage II. Furthermore, all DENV-2 strains analyzed presented an asparagine (N) in E390, previously identified as a probable genetic marker of virulence observed in DHF strains from Asian origin. The percentage of identity of the latter with the Dominican Republic strain isolated in 2001 combined to the percentage of divergence with the strains first introduced in the country in the 1990s suggests that those viruses did not evolve locally but were due to a new viral Lineage introduction in the country from the Caribbean.


Introduction
Dengue viruses (DENV) are the most important human arboviruses worldwide, transmitted by mosquitoes of the genus Aedes, Aedes aegypti is the main vector. Explosive epidemics have become a public health problem, economic impact, socially and politically significant [1,2].
Currently it is estimated that 70 to 500 millions dengue infections occur annually in 124 endemic countries. Nearly 3.6 billion people (55% of world population) are at risk of contracting the disease (DVI). The rapid global spread of DENV in the last 50 years resulted in the dispersal of genotypes associated with increased severity [3].
The four serotypes (DENV-1, DENV-2, DENV-3 and DENV-4) are closely related yet antigenically distinct and contain a positive-sense RNA genome that is translated as a single polyprotein and post-translationally cleaved into three structural proteins, capsid (C), premembrane (prM) and envelope (E), and seven nonstructural proteins, NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5. The RNA genome is packaged in an icosahedral capsid, and the nucleocapsid is surrounded by a lipid bilayer containing the E and M proteins [4,5].
DENV infection causes a spectrum of clinical disease ranging from an acute debilitating, self-limited febrile illness -dengue fever (DF) -to a life-threatening syndrome -dengue hemorrhagic fever/ dengue shock syndrome (DHF/DSS) [6]. Despite the similar disease manifestations, the DENV are genetically diverse with approximately 40% of amino acid sequence divergence. Distinct DENV genotypes can be characterized when the genetic divergence are higher than to 6% [7].
A recent analysis of 1,827 complete E gene sequences supported the existence of six genotypes for DENV-2: Asian genotype I, Asian genotype II, Southeast Asian/American genotype, Cosmopolitan genotype, American genotype and the Sylvatic genotype, the most genetically distinct genotype. Furthermore, the Southeast Asian/American genotype's topologies suggested a spatial division of this genotype into two major subclades [8].
In the Americas, the first DHF epidemics in the 80's were due to the introduction of the Southeast Asian/American genotype which replaced the American genotype and more severe cases with higher viremia were reported [9][10][11].
In Brazil, the disease has become a public health problem with explosive epidemics after the introduction of DENV-1 in 1986 in Rio de Janeiro [12]. However, the first DHF/DSS cases were only reported after the DENV-2 introduction in 1990 in the country [13,14]. From 1990 until the 26th epidemiological week of 2010, a total of 5,481,921 cases, including 17,203 cases of dengue hemorrhagic fever (DHF) and 1954 deaths were reported in the country [15].
Aiming to perform the phylogeny of the DENV-2 and its impact in the disease severity during 20 years of viral activity in Brazil, strains isolated from DF, DHF/DSS and fatal cases occurred since its introduction in 1990 until 2010, were analyzed. In this scenario, the partial sequencing (C/prM/M/E genes) of 25 DENV-2 strains was performed. To determine whether the evolutionary relationships observed for the C/prM/M/E genes were applicable to the complete genome, we further fully sequenced the coding regions of nine DENV-2 strains. In order to avoid mutations introduced by in vitro passages of the virus in cell cultures we used DENV-2 strains extracted directly from serum or originally isolated from cell cultures.

Ethical statement
The strains analyzed in this study belong to a previouslygathered collection from the Laboratory of Flavivirus, IOC/ FIOCRUZ, Rio de Janeiro, Brazil, obtained from human serum through the passive surveillance system performed by the Laboratory from an ongoing Project approved by resolution number CSN196/96 from the Oswaldo Cruz Foundation Ethical Committee in Research (CEP 274/05), Ministry of Health-Brazil. Samples were chosen anonymously, based on the laboratorial results and clinical manifestations input on the Laboratory database.

Viral strains
Viral strains consisted of DENV-2 (n = 34) isolated during epidemics occurred from 1990 to 2010 in six states in Brazil (Table 1). Each sample was accompanied by identification form containing clinical and epidemiological data. All strains were determined as DENV-2 serotype by reverse transcriptase polymerase chain reaction (RT-PCR) and or/virus isolation from DF (n = 19), DHF (n = 3), DSS (n = 1) and fatal cases (n = 4; 1 from DF, 2 from DHF and 1 with no classification available). Seven cases were not classified due to data unavailability.

RNA extraction
Viral RNA was extracted from infected cell culture supernatant or directly from the patients' serum using QIAamp Viral RNA Mini kit (Qiagen) following the manufacturer's instructions and stored at 270uC for DENV typing and sequencing.

RT -PCR (Reverse transcriptase-polymerase chain reaction)
RT-PCR for detecting and typing DENV was performed as described previously [16]. Briefly, consensus primers were used to anneal to any of the four DENV types and amplify a 511-bp product in a reverse transcriptase-polymerase reaction. A cDNA copy of a portion of the viral genome was produced in a reverse transcriptase reaction. After a second round of amplification (nested PCR) with type-specific primers, DNA products of unique size for DENV-2 (119 bp) were generated.

Dengue virus isolation
Virus isolation was performed by inoculation into C6/36 Aedes albopictus cell line [17] and isolates were identified by indirect fluorescent antibody test (IFAT) using serotype-specific monoclonal antibodies [18]. Briefly, patients' sera were inoculated into C6/ 36 Aedes albopictus cell monolayers in L-15 Medium (Leibovitz, Sigma) supplemented with 2% fetal calf serum (FCS, Invitrogen) and 0.2 mM of nonessential amino acids (Invitrogen). Cells were incubated at 28uC for 5 to 7 days and observed for cytopathic effects. Infected supernatant was clarified by centrifugation and virus stocks stored in 1-mL aliquots at 270uC until use.

Sequencing
Reverse transcription (RT) was performed using 5 mL of extracted RNA in 25 mL of AccessQuick RT-PCR System (Promega Corporation) and specific oligonucleotides primers (Table 1). To amplify the C/prM/M/E region of 2,325 bp, specific primers (1 to 4) were used to produce 4 overlapping amplicons of approximately 900 bp and to amplify the complete coding region (10,173 bp), 15 overlapping amplicons of approximately 900 bp (1 to 15). Thermocycling conditions consisted of a single step of 42uC for 60 minutes and 40 cycles of denaturation at 94uC (30 seconds), annealing at 56uor 63uC (60 seconds) depending on the set of primers, extension at 72uC (2 minutes) and a final extension at 72uC (10 minutes). Amplification was conducted using a Model 9700 thermal cycler (Applied Biosystems). PCR products were purified from 1.0% agarose gels using QIAquick Gel extraction Kit or QIAquick PCR purification Kit (Qiagen) and used as template for cycle sequencing. Sequencing reactions were performed as recommended in the BigDye Dideoxy Terminator sequencing kit (Applied Biosystems) and the products were analyzed using an automated 3130 DNA Sequencer (Applied Biosystems). Partial sequences (C/prM/M/E) and complete coding sequences for the unprocessed polyprotein (59 and 39 noncoding regions excluded) were deposited in GenBank (Table 2).

Author Summary
In Brazil, the first dengue haemorrhagic cases were reported after the DENV-2 introduction in Rio de Janeiro, which spread to other states in the country. Aiming to perform the molecular characterization and phylogenetic analysis of DENV-2 during twenty years of viral activity in the country, strains isolated from patients presenting different disease manifestations were sequenced. Phylogeny characterized the DENV-2 as belonging to the Southeast Asian genotype, however a distinction of two Lineages within this genotype has been identified. Furthermore, all strains presented an asparagine in E 390 , previously identified as a probable genetic marker of virulence. The results show a temporal circulation of genetically different viruses in Brazil, probably due to the introduction of a new viral lineage from the Caribbean, which lead to the re-emergence of this serotype after 2007, causing the most severe epidemic already described in the country.

Sequences and phylogenetic analysis
The analysis of similarities, percentage of identity and divergence among the strains analyzed were performed using Megalin Program (DNAstar, www.dnastar.com). The multiple alignment was performed using CLUSTAL W (http://www.ebi. ac.uk/clustalw/) and the phylogenetic analysis by MEGA 4 software (www.megasoftware.net), using the Maximum Likelihood method (ML), according to the Tamura-Nei model, with a bootstrap of 1,000 replications. Strains representative from the five genotypes available in Genbank (www.ncbi.nlm.nih.gov) were used for the comparison, DENV-1 (GenBank accession number GU370049), DENV-3 (accession number EF629369), and DENV-4 (accession number AF289029) strains were used as outgroup to root the trees (Table 3).

Results
In this study, the strains BR64022/98 isolated in the 90's and Jamaica 1983 were considered as reference strains for comparison purposes. The percentage of similarity among the 25 DENV-2 strains ranged from 80.3 to 99.9% when those compared to each other and to strains representative of the different genotypes available on GenBank. The partial genome sequencing analysis characterized the Brazilian DENV-2 strains from this study as belonging to the Southeast Asian genotype, however a distinction of two Lineages within this genotype has been identified. It was observed that strains circulating prior DENV-2 emergence (1990-2003) belong to Southeast Asian genotype, Lineage I and strains isolated after DENV-2 emergence in 2007 belong to Southeast Asian genotype, Lineage II (Figures 1 and 2). Furthermore, the latter were more closely related to strains from the Dominican Republic (DR59/01), representative from the Southeast Asian genotype, Lineage II.
When the 25 DENV-2 strains were compared to the strain BR64022/98, amino acid substitutions leading to change in the biochemical properties were observed on the C and prM genes. On the E gene, a total of twelve substitutions were observed, with nine resulting in a change on the amino acid change of biochemical property (Supplementary material 1). No consistent differences were observed on the E gene from strains isolated from cases with different clinical manifestations analyzed, suggesting that if the disease's severity has a genetic origin, it is not only due to the differences observed on the E gene.
To determine whether possible amino acids differences on other genes were related to disease severity, we fully analyzed (coding region) DENV-2 strains (n = 9), representative of DF cases isolated from 1990 to 1999 and strains isolated from fatal cases occurred after the DENV-2 re-emergence after 2007 until 2010. The strain 0450/2008, representative of the DENV-2 reemergence isolated from a DF secondary case who evolved to death was fully sequenced and its comparison to the strain from the Dominican Republic (DR59/2001), representative of the DENV-2 re-emergence, showed 22 amino acid substitutions. Likewise, the strain 0690/2008 isolated from a DHF case occurred also during the re-emergence of DENV-2 had nine had amino acid substitutions when compared to the strain DR59/2001, with seven of those leading to amino acid biochemical property change (Table S1).
The DENV-2 strain 0337/2008 isolated from a newborn presenting a high anti-DENV IgG titer who evolved to death, infected probably due transplacental transmission as his mother was diagnosed with acute DENV infection, showed substitutions on NS2A, NS4A and NS5, which were shared with the other two strains isolated from fatal cases (Table S2). The results obtained by the DENV-2 full-length genome sequencing did not point out consistent differences related to a more severe disease.
A substitution on E 390 (NRD) was reported as resulting in a reduction in viral replication in macrophages and dendritic cells [19] whereas E 390 (DRN) resulted in enhanced replication, maturation and activation of macrophages, enhancement of the immune response with an increased production of cytokines, increased vascular permeability and consequently a greater chance of developing DHF [20].All DENV-2 strains analyzed presented an asparagine (N) in E 390 , previously identified as a probable genetic marker of virulence observed in DHF strains from Asian origin.
The percentage of identity of the re-emergent DENV-2 with the Dominican Republic strain isolated in 2001 combined to the percentage of divergence with the strains first introduced in the country in the 90's suggests that those viruses did not evolved locally but were due to a new viral Lineage introduction in the country from the Caribbean.

Discussion
In the Americas, the first DENV-2 was isolated in 1953 in Trinidad [21] and the first DHF epidemic caused by this serotype occurred in Cuba in 1981 after the introduction of DENV-2 genotype originated in Southeast Asia [10,22]. Epidemics studies showed that the DENV-2 introduced in Brazil, Colombia, Venezuela and Mexico had a common ancestor with isolates from Southeast Asia, suggesting the direct transmission from that region to the Americas [23].
In the present study, the analysis based on the sequencing of the C/prM/M/E genes (2,325 bp) from 25 DENV-2 Brazilian isolates divided those strains in two distinct groups, one formed by DENV-2 isolated from 1991 to 2003 and another with strains isolated from 2007 to 2010 following the re-emergence of this serotype in the country. Corroborating previous phylogeny [26][27][28][29] strains isolated from 1991to 2003 were classified as Southeast Asian genotype, Lineage I and presenting similarities with the Brazilian strain BR64022/98 and the strain Jamaica/83. However, the strains isolated between 2007 and 2010, showed higher similarity with the strain DR59/01, from the Dominican Republic, representing the Southeast Asian genotype, Lineage II, corroborating the analysis by Oliveira et al [32]. A study by Aquino et al [33] demonstrated that DENV-2 strains from Paraguay could also be grouped into two distinct lineages within the Southeast Asian genotype and suggested the introduction of a new lineage possibly associated a serotype shift from DENV-3 to DENV-2, as observed in Brazil in 2007 and 2008 [31].
The absence of DENV-2 circulation in the years prior to its reemergence and the high similarity observed between those viruses and the strain isolated in the Dominican Republic in 2001, suggests the introduction of a new lineage of DENV-2 causing the 2008 epidemic in Brazil. Romano et al [34] also demonstrated that DENV-2 strains isolated in Sao Paulo State in 2010 were in a monophyletic group with the strains circulating in Rio de Janeiro in 2007 and 2008 and that those were closely related to strains isolated in Cuba and Dominican Republic, with a small genetic distance, suggesting that this new lineage of DENV-2 re-emerged in of Brazil may have been imported the Caribbean. Although genetic variants of DENV have been implicated in disease severity in the past [35,36], it was with the advance of evolutionary studies based on phylogenetic analysis combined to epidemiological data that genotypes within the distinct serotypes were associated with a greater or lesser disease severity [11,[37][38][39][40].
The strain isolated from a DHF case in 2000 (strain RJ/67922/ 2000) presented an exclusive substitution on prM 143 (TRI) when compared to the other strains analyzed in this study. However, substitutions related to DHF/DSS cases were identified on prM 16 and prM 81 [41].
Substitutions were found on the residues E 129 (VRI) and E 131 (LRQ), and these are related to the division of the Southeast Asian genotype in two distinct clades, corroborating the observations that amino acids on E 129 and E 131 are in critical markers for genetic classification of DENV [33,42].
All 34 strains analyzed in this study presented an asparagine (N) on E 390 , previously characterized as a probable trigger for DHF detected in strains of Asian origin [43]. Mutations on the flaviviruses domain III of E protein can induce virulence or attenuation of the virus to escape from the immune system [44,45] and in this study, changes were observed throughout this domain (aa 297 to 394). The DHF case, which culminated in death (59382/1997) showed amino acid differences only in  [46] demonstrated that the intracellular production of M ectodomain of all four DENV serotypes of DENV induce apoptosis in host cells. The carboxy terminus of prM protein with nine amino acids (aa 32-40) of some flaviviruses was designated as Apopto M [46] and appears to play an important role in inducing apoptosis and cytopathic effects [46][47][48].
Several changes were observed along the NS protein genes. Studies conducted by Yábar, [49] show that mutations in NS1 are related to the development of DHF/DSS cases when they were compared to patients with DF.
Despite the functional importance of mutations in NS genes remains unknown, future studies can elucidate their role in the emergence of strains and/or pathogenesis of the disease. It was not possible to correlate the role of Lineage II emergence with an increased severity of cases observed in the period between the years 2007-2010. Furthermore, the occurrence of secondary infection may have been the risk factor for the development of more severe cases.
In conclusion, this result shows a temporal circulation of genetically different viruses in Brazil probably due to the introduction of a new viral lineage from the Caribbean which lead to the re-emergence of this serotype after 2007. In 2007-2008, DENV-2 was responsible for most severe epidemic already described in the country, with 787,726 cases reported and 491 deaths [31]. Moreover, the Caribbean has been suggested as an important region for the circulation of DENV-2, importation and exportation of strains from and to Central America and South America [42,50,51].
In the past 20 years, DENV-2 activity in Brazil has contributed significantly to changes in the disease morbidity and sudden age shift [30]. In dengue endemic countries, displacement of DENV serotypes, genotypes and lineages have been reported previously and have been associated with changes in the disease severity [40,[52][53][54][55]. This emphasizes the need of straightening virological surveillance to monitor the emergence or re-emergence of DENV strains with pathogenic potential to cause epidemics.

Supporting Information
Table S1 Molecular characterization of DENV-2 strains isolated in Brazil based on the partial genes analysis. (DOCX)