Dengue

Dengue Bulletin Volume 28 (2004)

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Molecular Characterization of Brazilian Dengue Viruses

Marize Pereira Miagostovich^#, Flávia Barreto dos Santos and Rita Maria Ribeiro Nogueira

Laboratory of Flavivirus, Department of Virology, Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Fiocruz Avenida Brasil 4365 Manguinhos, Rio de Janeiro 21040-360, RJ, Brazil

Introduction

The dengue (DEN) virus belongs to the Flaviviridae family, genus Flavivirus,and it has four distinct antigenic serotypes (1 to 4) that cause a spectrum of diseases ranging from asymptomatic, mild, undifferentiated fever and classic dengue fever (DF) to more severe forms known as dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS)^[1,2].

The DEN virus is a spherical particle of approximately 500 Å in diameter, lipid enveloped that includes one segment of a single-stranded positive sense RNA with ~11,000 nucleotides in length. The genomic RNA contains a single long open reading
frame (ORF) of over 10,000 nucleotides that encodes a polyprotein precursor of about 3,400 amino acid residues which is co- and post-translationally processed by the host cell and virus-specific protease to yield structural and nonstructural proteins. The coding protein region starts with the sequence for the core (C), precursor of membrane (prM/M) and the envelope (E) structural proteins, followed by a series of seven nonstructural (NS) proteins ordered as follows: NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5. The ORF is flanked by two untranslated regions (5’ and 3‘ UTR) and has a type I cap at its 5’ end (m7GpppAmp) and appears to lack a 3’-terminal poly A tract^[3,4].

Electron micrographs show that the virion is characterized by an electron dense core that consists of an isometric nucleocapsid, made up of a single C (100 amino acids) protein, surrounded by a double lipid layer, whereas both E (495 amino acids) and M (75 amino acids) proteins are associated^[5]. E-glycoprotein is the major surface protein and as showed by crystallography, the flat elongated dimmer extends parallel to the viral membrane^[6]. The E protein is associated with a number of biological activities, being the most important antigen with regard to virus biology and immunity^[7].

The intratypic variation of DEN was demonstrated by the fingerprinting method that determined genetic variants within each serotype and employed the term topotype to define variants representing samples from the same geographical region^[8]. From 1990 onwards, the molecular analysis by the partial sequencing of the DEN genome gathered the topotypes in genomic groups (genotypes) and became an important tool to determine their genetic variation and to identify risk factors associated with the transmission of particular strains^[9].

The E gene has been the most commonly surveyed gene in dengue molecular epidemiology^[10-15], although genes that encode nonstructural proteins and non-coding regions have also been used in the phylogeny studies^[16-22].

Once intratypic variations among different serotypes were associated with the disease severity, technologies for the molecular characterization of DEN became an indispensable tool for laboratories performing virological surveillance programmes. This paper presents the most relevant results of the molecular characterization of DEN strains isolated in Brazil during the last 18 years since dengue became endemic and surveillance programmes were implemented in the country.

DEN-1

The first molecular analysis for DEN Brazilian strains characterization was performed by an analysis on genome fragments from DEN-1 by using restriction endonuclease (RE) enzymes. In this study, restriction fragment heterogeneity by Hae III digestion of cDNA products was used to map the distribution of DEN-1 topotype found in the American region. The strains isolated in the state of Rio de Janeiro from human serum specimens from 1986 to 1994^[23,24] were grouped in the American (Caribbean) topotype, recognized as the only one circulating in the Americas^[8,25]. The percentage of the similarity observed among DEN-1 Brazilian strains ranged from 60% to 94%, showing the evolution of those samples since its introduction in the state in 1986.

By the sequencing of 240-nucleotides (nts) spanning the E/NS1 junction (111 nts from the 3’ end of E gene and 129 nts from the 5’ end of NS1 gene), the DEN-1 Brazilian strains were classified as belonging to genotype I which comprises of strains from the Americas, Africa and South-East Asia^[9].

The DEN-1 Brazilian strains were also analysed by using one-step amplification with four primers that target regions spanning polymorphic endonuclease restriction specific sites (RSS-PCR) and all of them were grouped into subtype C, which corresponds to the largest genotypic group of DEN-1 described as genotype 1^[9,26]. The RSS-PCR has become an alternative tool routinely used, which has allowed the characterization of DEN strains for molecular epidemiological studies performed in endemic countries, providing rapid identification of viruses currently circulating^[27-30].

Another study performed with the DEN-1 Brazilian strains compared the complete sequences of three strains isolated in 1990, 1997 and 2001. The genome analysis of those strains revealed a remarkable conservation of the structural proteins and 27 amino acids substitutions in the nonstructural genes, and 12 of them in the NS4B-NS5 and nine specific to strains BR/97 and BR/01. Those findings also suggested that recombinant events might have occurred, since some amino acids substitutions were previously identified in DEN-1 strains sequenced so far^[31]. The evidence that the genetic diversity of DEN might be generated by recombination among those viruses has been described^[32-34].

DEN-2

The DEN-2 fingerprinting analysis of the American strains showed that this serotype exists in the American continent as two topotypes representing strains from the Caribbean region (Puerto Rico) and from the Americas, India and South Pacific^[8]. By the sequencing of the E/NS1 junction those topotypes could be related to genotype I (Native American) and to genotype III (South-East Asian/American), respectively. This latter genotype was introduced into the Americas in 1981, and was responsible for the first DHF/DSS epidemic that occurred in the continent and spread throughout the region over the next two decades^[35,36]. The direction of the transmission from South-East Asia to the Americas was demonstrated as well, since DEN-2 from Brazil, Colombia, Mexico and Venezuela have a common progenitor with those from South-East Asia^[36].

In Brazil, the first molecular characterization of DEN-2, introduced in the country in 1990^[37,38], was performed by RE analysis and showed a similarity of 80% with the 1981 Jamaica isolate, suggesting the spread of those viruses from the Caribbean region to South America^[25].

The geographical origin of DEN-2 Brazilian strains was also established by the direct sequencing of cDNA fragments amplified by the polymerase chain reaction of a fragment encoding amino acids 29 to 94 in the E gene. Considering a divergence of 6% between the nucleotide sequences as a cut-off for genotype classification, it was demonstrated that the Brazilian strains belonged to the South-East Asian/American genotype. The comparison of the three DEN-2 strains isolated in Rio de Janeiro, two of them obtained from classic dengue cases and one from a fatal case, did not identify the markers for virulence in the region studied^[10].

The analysis of DEN-2 samples isolated in the states of Rio de Janeiro, Ceará, Bahia, and Alagoas between 1990 and 1995 was performed by the partial sequencing of nts 1685 and 2504 encompassing the E gene and demonstrated the spread of this serotype from Rio de Janeiro to other states^[39].

All the characteristics observed in the Brazilian DEN-2 genotype were confirmed by the full-length analysis of the nucleotide and amino acids sequence (GenBank access # AF489932)^[40]. The Asian-specific non-conserved amino acid differences, previously described by Leitmeyr et al.^[41] as well as additional differences specific to the Brazilian strain were found in E, NS3, and NS5 genes^[40]. Changes in the E protein could affect the immunogenicity or cell entry/tropism, whereas changes in NS3 (helicase/protease) and NS5 (RNA-dependent RNA polymerase) could affect replication efficiency. In addition, differences in the predicted secondary structure of the 5’ and 3’ untranslated regions were found between the South-East Asian/American and native American genotypes; in these regions, the Brazilian isolate was identical to the South-East Asian/American strains in sequence and consequently in the predicted secondary structures^[40]. These similarities with the South-East Asian/American genotype were also reported recently for the Martinique 703/98 strain after a complete analysis of the genome^[42]. 

In Brazil, this DEN-2 genotype was responsible for some clinical features, mainly related to the severity of the disease. In regions where DEN-2 accounted for primary infections, as in the states of Bahia and Espirito Santo^[43], the most common clinical feature consisted of classic fever, with frequent exanthema, pruritus and a few severe cases. However, in other states where DEN-2 circulated after extensive epidemics caused by DEN-1, as in Rio de Janeiro, Ceará, Pernambuco and Rio Grande do Norte, an increase in the number of severe cases was observed. The first DHF/DSS case was reported in Rio de Janeiro after the introduction of DEN-2 in 1990, and it was accompanied by an increasing number of hospital admissions resulting from DEN-2 secondary infections^[44,45].

By using RSS-PCR we were able to analyse geographically and temporally distinct Brazilian DEN-2 strains encompassing ten years (1990 to 2000). The analysis of the RSS-PCR products showed that all Brazilian strains presented the same pattern, presenting consistent and reproducible amplicons of 582bp and 100bp and, occasionally, extra amplicons of 676 bp or 150 bp^[29]. The DEN-2 Brazilian RSS-PCR pattern was consistent; however, it did not match any of the RSS-PCR patterns previously described by Harris et al.^[46] Once the method was developed with DEN-2 isolates obtained from 1964-1986, the ongoing evolution of those viruses over the last 15 years could explain the genetic diversity observed. Despite those observations, the sequence of the E/NS1 gene junction (GenBank Access # AF529064 to AF529078) showed that the Brazilian DENV-2 strains still belonged to the South-East Asian/American genotype^[29].

DEN-3

Different from the DEN-3 genotype IV (topotype Caribbean) responsible for epidemics in the ‘60s and ‘70s, the DEN-3 re-introduced in the American continent after an absence of 17 years belonged to genotype III (topotype Sri Lanka), represented by strains from Sri Lanka and India, which are associated with DHF/DSS cases in those countries^[13,47].

By the time of DEN-3 isolation in Brazil^[48], RSS-PCR was extremely valuable which once allowed the rapid characterization of the first strain as subtype C, confirming the introduction and direction of transmission of those viruses from Central to South America^[28,46].

After the RSS-PCR analysis, the nucleic acid sequencing from positions 278 to 2550 of DEN genome was performed (Access number AY038605) and the parsimony analysis generated a phylogram assigning a Brazilian DEN-3 strain to genotype III, reconfirming those data^[13,28]. The similarity rate of a Brazilian DEN-3 strain to others represented by the same subtype III ranged from 96% to 98% and 98% to 99% for nucleic acid and deduced amino acid sequences, respectively. The comparison of the Brazilian DEN-3 with strains isolated in Guatemala showed a total of 14 nucleic acid substitutions, with one of them resulting in an amino acid change from histidine to arginine^[28].

As a result of several DEN-3 epidemics in Latin American countries, a large number of DEN-3 genome sequences have been recently deposited in the GenBank^[49-51]. A phylogenetic study compromising DEN-3 strains isolated in Sri Lanka, East Africa and Latin America confirmed the establishment of the new DEN-3 genotype^[51]. According to the author, there are two separate lineages formed within genotype III: Group A consisting of isolates from 1981 to 1989 in Sri Lanka and Group B which was expanded in three distinct clades including isolates from 1989 to 1998 in Sri Lanka, strains isolated in East Africa from 1985 to 1993 and isolates from 1994 in Latin America. The phylogenetic analysis suggested that genotype III was introduced from the Indian subcontinent into East Africa in the 1980s and from Africa into Latin America in 1994, showing a single genotype introduction in the continent and its subsequent diversification^[52]. In the same year, Peyrefitte et al.^[53] showed a high similarity between the DEN-3 Martinique and the Brazilian strains. Furthermore, the complete genome characterization of the Brazilian DEN-3 sequence (AY679147) strain confirmed an insertion of 11 nts in the 5´ non-coding region of the genome as previously described for the Martinique strain^[53].

The severity of the disease and the occurrence of deaths resulting from primary infections during the DEN-3 epidemic in the state of Rio de Janeiro in 2002 could be explained partially by the virulence of this particular genotype^[28,54]. Fatal cases, resulting from primary dengue infections, were previously described^[55] before the DEN-3 genotype III introduction in Brazil. However, the highest number of DHF/DSS cases that occurred in the state were due to secondary infection by the South-East Asian/American DEN-2 genotype^[45]. Those findings corroborated the previous observations that some DEN strains can be more virulent than others, representing an important risk factor for DHF/DSS and that antibody-dependent enhancement (ADE) itself does not explain all cases of severe disease^[51,56-58]. Recently, it was suggested that the more virulent genotypes were now replacing those that had a lower epidemiological impact throughout the world^[59].

Conclusion

In the last few decades, dengue has spread as a pandemic in the American continent, starting in the Caribbean islands and expanding to North, Central and South America^[60]. In this context, the dengue epidemiological profile in Brazil has changed from a non-endemic to a hyperendemic one. Since the 1980s when the first DEN strains were isolated, more than 3 million dengue cases and nearly 2,090 DHF/DSS cases have been reported in the country (www.funasa.gov.br)^[61,62].

The endemicity of dengue in 25 out of the 27 federative units, the remarkable virulence of the DEN-2 and DEN-3 genotypes and the risk of the introduction of DEN-4 in the country highlight the alarming dengue epidemiological picture in Brazil. In this scenario, use of rapid methods for DEN identification and molecular characterization are indispensable tools in the virological surveillance laboratories, mainly due to an obvious need to characterize dengue genotypes before a major outbreak occurs^[26,46,63]. The partial sequencing of DEN strains genome has also been used routinely for DEN molecular epidemiological studies, and it recently characterized the co-infecting genotypes of DEN-1 and DEN-2 in a patient presenting classic dengue fever in São Paulo^[64].

The knowledge of the virus genotype circulating in a particular region has also implications for the potential introduction of vaccines, allowing the evaluation of the genomic relations between the viruses used in vaccine development and the circulating strains. The ability of pre-existing dengue antibodies to neutralize better certain DEN variants than others has been demonstrated. Some strains may produce a more severe
disease, not because of the virulence-inherited properties but because antibodies from a primary infection may enhance infection with one genotype while neutralizing infection with a distinct one^[52,65].

Given the limited options available for dengue control, active surveillance programmes with continuous monitoring of dengue infection in communities is still one of the strategies available to detect the introduction of new serotypes/genotypes, and, consequently, to prevent the occurrence of epidemics, thus minimizing the impact of the circulating strains.

Acknowledgements

To Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ), Fundação Oswaldo Cruz (FIOCRUZ), PAPES III – FIOCRUZ. Thanks are also due to the staff of the Laboratory of Flavivirus for their technical assistance.

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