JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 2011, p. 3370–3374 0095-1137/11/$12.00 doi:10.1128/JCM.00950-11 Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Vol. 49, No. 9

Molecular Epidemiology of Autochthonous Dengue Virus Strains Circulating in Mexico䌤 Pilar Rivera-Osorio,1 Gilberto Vaughan,2 Jose Ernesto Ramı´rez-Gonza´lez,3 Salvador Fonseca-Coronado,4 Karina Ruı´z-Tovar,2 Mayra Yolanda Cruz-Rivera,5 Juan Alberto Ruı´z-Pacheco,2 Mauricio Va´zquez-Pichardo,1 Juan Carlos Carpio-Pedroza,2 Fernando Ca´zares,2 and Alejandro Escobar-Gutie´rrez2* Departamento de Virología, Instituto de Diagno ´stico y Referencia Epidemiolo ´gicos, Secretaría de Salud, Mexico City, Mexico1; Departamento de Investigaciones Inmunolo ´gicas, Instituto de Diagno ´stico y Referencia Epidemiolo ´gicos, Secretaría de Salud, Mexico City, Mexico2; Departamento de Biología Molecular, Instituto de Diagno ´stico y Referencia Epidemiolo ´gicos, Secretaría de Salud, Mexico City, Mexico3; Unidad de Investigacio ´n Multidisciplinaria, Facultad de Estudios Superiores Cuautitla ´n, Universidad Nacional Auto ´noma de Me´xico, Cuautitla ´n-Izcalli, State of Mexico, Mexico4; and Departamento de Microbiología y Parasitología, Facultad de Medicina, Universidad Nacional Auto ´noma de Me´xico, Ciudad Universitaria, Mexico City, Mexico5 Received 9 May 2011/Returned for modification 28 June 2011/Accepted 1 July 2011

Dengue virus (DENV) is the most important arthropod-borne viral infection in humans. Here, the genetic relatedness among autochthonous DENV Mexican isolates was assessed. Phylogenetic and median-joining network analyses showed that viral strains recovered from different geographic locations are genetically related and relatively homogeneous, exhibiting limited nucleotide diversity. strains have also been identified to be circulating in the country, a phenomenon that has important implications regarding changes in genetic variation and antigenic makeup (17). Dengue viruses are enveloped, positive-polarity, singlestranded RNA viruses belonging to the Flavivirus genus and

Dengue virus (DENV) is the most important mosquitoborne human viral disease and a very important public health problem in diverse regions of Asia, the Pacific, Africa, and the Americas (10). In Mexico, circulation of all four DENV serotypes has been reported (3, 6, 8, 16). Moreover, recombinant

FIG. 1. Sliding window and entropy analysis. (A) Sliding window and TOPD analysis. The graphic represents the similarities between all individual subtrees against the reference tree (entire DENV E protein gene). (B) Nucleotide variability analysis. ␲ was calculated by individual windows and plotted against nucleotide position.

* Corresponding author. Mailing address: Departamento de Investigaciones Inmunolo ´gicas, Instituto de Diagno ´stico y Referencia Epidemiolo ´gicos, Carpio 470, Mexico City 11340, Mexico. Phone: (52) 55-53427563. Fax: (52) 55-5341-3264. E-mail: [email protected]. 䌤 Published ahead of print on 20 July 2011. 3370

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FIG. 2. Study sites. The communities from which dengue virus isolates were recovered are indicated on the map. Samples were collected in either 2006 (white circles) or 2007 (black circles) from DF (single circles) and DHF (double circles) cases identified in 22 different states in Mexico.

the Flaviviridae family. Four antigenically different viral serotypes have been recognized so far which do not provide cross immunity upon natural infection. Likewise, several evolutionary and molecular studies based on the nucleotide sequence of the gene coding for the envelope (E) protein have shown that dengue viruses can be clustered into different genotypes within each individual serotype (12, 20, 21). It has been proposed that the characteristic immunopathogenesis of DENV infections is the result of an enhanced immune response developed during the course of a secondary infection with a heterologous viral serotype (9, 14). However, the existence of virulent genotypes with increased replication rates suggests that the viral strain itself might also play an important role in disease outcome (1). Furthermore, some genotypes have been associated with the occurrence of dengue hemorrhagic fever (DHF) epidemics, while some others have been linked mostly to relatively mild clinical manifestations (19). Thus, the circulation of certain genotypes might increase the risk for the development of DHF cases. Therefore, molecular surveillance is required in areas where DENV is endemic to accurately monitor the introduction of new more aggressive viral strains. The goal of this study was to assess the genetic relatedness among autochthonous DENV Mexican isolates circulating in

the country between 2006 and 2007. The usage of small subgenomic regions has largely been recommended to conduct molecular epidemiological outbreak investigations and relatedness studies of different viral diseases (22, 24). Here, initially a relatively small region (⬃473 nucleotides [nt] in length) capable of reflecting the nucleotide complexity along the entire coding region of the E protein was identified to conduct molecular analysis of DENV strains. For this, a comprehensive nucleotide sequence alignment based on the entire coding region of the E protein, including all known DENV serotype 1 (DENV-1) Mexican strains, currently available from GenBank (http://www.ncbi.nlm.nih.gov/GenBank/index.html), was generated and scanned using a sliding window approach (window size, 500 nt; step size, 10 nt). Subsequently, each individual window was used to create the corresponding matrix distance (Kimura 2-parameter) and tree (Newick tree format) using the neighbor-joining method as implemented in PHYLIP v3.5 software. Afterwards, the differences between each individual Newick tree and a reference tree (whole E protein sequences) were calculated using the nodal method as implemented in TOPD/FMTS software (18). Using this approach, a region located along domain III of the DENV E protein was shown to better resemble the topology of the reference tree (Fig. 1A). The nucleotide diversity (␲) along the entire E protein was

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also calculated using DnaSP v5 software (15). This analysis showed a rather flat line throughout domain III of the E protein, indicating that nucleotide variability was rather homogeneous all along the gene coding for the E protein (Fig. 1B). Thus, a set of primers (forward, 5⬘-GACTGGGGCGACAGA AATCC-3⬘; reverse, 5⬘-AAGTCCCATGCGGTGTCTCC-3⬘), situated between nucleotides 1727 and 2200, capable of amplifying the region of interest were designed, and 100 different DENV-1 strains, the most common serotype circulating in Mexico between January 2006 and December 2007, were randomly selected. All specimens belonged to acute cases of dengue virus from different regions of DENV endemicity in Mexico with no history of recent domestic or international travel (Fig. 2). The samples were inoculated into C6/36 cells for 7 days at 28°C and subsequently subjected to RNA extraction, PCR amplification, and sequencing. The vast majority of these cases (96) were primary infections. Only four cases were compatible with secondary infection, classified as such by their characteristic antibody profile. Thirty-four isolates were recovered from DHF cases, including three secondary infections, implying that most hemorrhagic manifestations were seen among individuals with primary infections. Partial molecular characterization of all isolates was conducted in order to identify molecular patterns associated with the prohemorrhagic viral strains. Multiple sequence alignment was performed using MUSCLE v3.8.31 software (7). Phylogenetic trees were generated from a comprehensive alignment that included all 44 unique nucleotide sequence patterns (UNSPs), using the neighbor-joining method (Kimura 2-parameter), and branch topology was verified by generating 1,000 bootstraps as implemented in MEGA4 software (23). Additionally, 47 Mexican isolate sequences (http://www.ncbi.nlm.nih.gov/GenBank /index.html), belonging to acute cases of dengue virus for which the year and place of collection were available, were also included in this analysis. Reference sequences belonging to all DENV-1 genotypes were included to root the tree. Sequence analysis showed that all DENV strains belonged to genotype III (Fig. 3). DHF cases, including only one secondary infection, were represented by 16 different haplotypes. Most DHF haplotypes (13) included a single isolate, while three haplotypes included multiple isolates (16, 13, and 2, respectively). These strains did not cluster together, but rather spread throughout the tree, or exhibit any distinctive molecular pattern within this subgenomic region. However, a nucleotide substitution, guanine instead of adenine, at nucleotide position 1820 was relatively common (33.3%) among viral strains associated with DHF compared to dengue fever (DF)-causing viruses (8.5%). This substitution did not lead to any phenotypic change (silent mutation), keeping the corresponding amino acid (lysine 575) as a conserved residue. Whether this nucleotide substitution

FIG. 3. Phylogenetic analysis. Phylogenetic analysis (neighbor-joining method) of all DENV-1 Mexican UNSPs based on sequences belonging to domain III of the DENV E protein gene. DF strains are represented by black squares, and DHF isolates are indicated by gray squares. DENV genotype III isolates collected during the 1980s and 1990s are indicated by shaded boxes.

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FIG. 4. Median-joining network analysis. Each node represents a unique haplotype. The size of the node reflects the frequency of each haplotype. Geographic locations are depicted in different colors. The main haplotypes (1, 2, 3, and 4) are indicated by their corresponding number.

plays role in strain virulence or is a marker, along with some other nucleotide substitutions, for prohemorrhagic viruses requires further studies. We hypothesize that the evolution of DENV, shaped primarily by negative selection (5) and characterized by infrequent recombination events (11), might be organized through coordinated nucleotide substitutions that can be viewed as systematic categories that can be built into a network. Our group is currently engaged in a nationwide study aiming to identify and characterize the interactions between nucleotide substitutions occurring at the whole-genome level in Mexican isolates. Median-joining network analysis showed four major nodes (main haplotypes) at the center of the network (Fig. 4). All 39 remaining nodes (minor haplotypes) were located around the main haplotypes. Haplotypes 1 and 2 were different by only one nucleotide; likewise, haplotypes 3 and 4 differed from each other by only one position. Haplotypes 2 and 3 presented three nucleotide substitutions. Haplotype 1 grouped 16 different isolates and was surrounded by 17 minor variants, of which 12 had only one different nucleotide, including haplotype 2. The 5 remaining strains presented 2 to 3 nucleotide differences. Similarly, most minor strains (7 strains) around haplotype 2, which compiled 20 different isolates, differed by 1 position, and only one presented 2 nucleotide differences. Five viral strains surrounding haplotype 3 (single isolate) presented between 3 and

4 nucleotide differences, and three more exhibited 1 nucleotide difference. For haplotype 4 (8 different isolates), all 5 minor variants differed by 1 nucleotide. Two variants, indicated by asterisks in Fig. 4, were not directly connected to any of the major haplotypes. These findings suggest that the DENV-1 population circulating in Mexico during the observation time has remained relatively stable, with no introduction of new lineages into the country. DENV genotypes featured distinctive geographic distributions in which some genotypes have been shown to be more cosmopolitan than others (12). In the particular case of DENV-3, genotype III has successfully spread to Africa, the Americas, and India, while genotype I has been largely restricted to Southeast Asia. Whether these differences in geographic distributions are the result of fitness differences remains to be determined. It has been postulated that both fitness and virulence might have a major effect on viral population structure (13). Thus, abrupt displacement of a particular lineage by more fit viral strains leads to changes in the genetic composition and architecture of the viral population. This disturbance of the structural arrangement of the coexistent viral strains in a given time might potentially modify the epidemiology of the disease. The best example of the occurrence of such events has been the replacement of the low-virulence DENV-2 American genotype by the more aggressive Southeast Asian genotype lineage (4).

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Recently, it has been postulated that multiple introductions of diverse viral lineages have been taking place in Mexico during the past few decades (2). However, cocirculation of different lineages at any given time point has been rather sporadic, suggesting that frequent lineage replacement is the force shaping the structure of the viral population circulating in the country. Our findings are in accordance with these previous observations; thus, during this 2-year observation period, a rather homogeneous viral population was identified in different geographic regions in Mexico. Based on these findings, one can suggest that in the future, new emerging viral lineages could potentially replace the viral strains that are currently established in the country, thus changing the structure and possibly the antigenic makeup of the viral population. This phenomenon could eventually lead to the introduction of viral strains with increased virulence. Therefore, molecular surveillance of DENV is of high importance and should be conducted rigorously to monitor the presence of new viral lineages in the country. This work was partially supported by Consejo Nacional de Ciencia y Tecnología (CONACyT), grant Salud-2008-C01-87653. S. FonsecaCoronado received funding from Programa de Formacio ´n e Incorporacio ´n de Profesores de Carrera en Facultades y Escuelas para el Fortalecimiento de la Investigacio ´n (PROFIP), DGAPA, UNAM. REFERENCES 1. Anderson, J. R., and R. Rico-Hesse. 2006. Aedes aegypti vectorial capacity is determined by the infecting genotype of dengue virus. Am. J. Trop. Med. Hyg. 75:886–892. 2. Carrillo-Valenzo, E., et al. 2010. Evolution of dengue virus in Mexico is characterized by frequent lineage replacement. Arch. Virol. 155:1401–1412. 3. Cisneros, A., et al. 2006. Dengue 2 genotypes in the state of Oaxaca, Mexico. Arch. Virol. 151:113–125. 4. Cologna, R., P. M. Armstrong, and R. Rico-Hesse. 2005. Selection for virulent dengue viruses occurs in humans and mosquitoes. J. Virol. 79:853–859. 5. Descloux, E., V. M. Cao-Lormeau, C. Roche, and X. De Lamballerie. 2009.

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