Genetic characterization of dengue virus type 3 isolates in the State of Rio de Janeiro, 2001

The genetic characterization of dengue virus type 3 (DEN-3) strains isolated from autochthonous cases in the State of Rio de Janeiro, Brazil, in 2001 is presented. Restriction site-specific (RSS)-PCR performed on 22 strains classified the Brazilian DEN-3 viruses as subtype C, a subtype that contains viruses from Sri Lanka, India, Africa and recent isolates from Central America. Nucleic acid sequencing (positions 278 to 2550) of one DEN-3 strain confirmed the origin of these strains, since genotype III - classified by sequencing - and RSS-PCR subtype C are correlated. This genetic subtype has been associated with hemorrhagic dengue epidemics and the information provided here could be useful to implement appropriate prevention and control measures.

Dengue virus type 3 isolation from Aedes aegypti in the municipality of Nova Iguaçu, State of Rio de Janeiro

In a prospective field study conducted from July 2000 to June 2001, adult Aedes aegypti and Ae. albopictus mosquitoes were caught from the municipality of Nova Iguaçu, State of Rio de Janeiro, Brazil. Virus isolation in Ae. albopictus clone C6/36 cell line and a semi-nested reverse transcription-polymerase chain reaction detected only dengue virus type 3 in three pools of Ae. aegypti, despite the co-circulation of DEN-1, DEN-2 and DEN-3 serotypes in that area. No viruses were detected in Ae. albopictus mosquitoes. This virological surveillance consists in a sentinel system alerting for dengue outbreaks.

Evidence for the co-circulation of dengue virus type 3 genotypes III and V in the Northern region of Brazil during the 2002-2004 epidemics

The reintroduction of dengue virus type 3 (DENV-3) in Brazil in 2000 and its subsequent spread throughout the country was associated with genotype III viruses, the only DENV-3 genotype isolated in Brazil prior to 2002. We report here the co-circulation of two different DENV-3 genotypes in patients living in the Northern region of Brazil during the 2002-2004 epidemics. Complete genomic sequences of viral RNA were determined from these epidemics, and viruses belonging to genotypes V (Southeast Asia/South Pacific) and III were identified. This recent co-circulation of different DENV-3 genotypes in South America may have implications for pathological and epidemiological dynamics.

Molecular characterization of Dengue viruses type 1 and 2 isolated from a concurrent human infection

In 2001, an autochthonous case of dual viremia, resulting from naturally acquired dengue virus DEN-1 and DEN-2 infections was detected during the dengue outbreak that occurred in Barretos, a city with about 105,000 inhabitants in the North region of São Paulo State. Serotype identification was based on virus isolation to C6/36 mosquito cells culture and immunofluorescence assays using type-specific monoclonal antibodies. The double infection was also confirmed by reverse transcriptase polymerase chain reaction (RT-PCR). Comparative analysis of the 240-nucleotide sequences of E/NS1 gene junction region between the genome of DEN-1 and DEN-2 isolates of the corresponding reference Nauru and PR 159S1 strains, respectively, showed some nucleotide differences, mainly silent mutations in the third codon position. Results of maximum likelihood phylogenetic analysis of E/NS1 gene sequences indicated that both genotypes of DEN-1 and DEN-2 viruses recovered from double infection in Barretos belonged to genotypes I and III, respectively.

Dengue virus type 3 in Rio de Janeiro, Brazil

Dengue virus type 3 was isolated for the first time in the country as an indigenous case from a 40 year-old woman presenting signs and symptoms of a classical dengue fever in the municipality of Nova Iguaçu, State of Rio de Janeiro. This serotype has been associated with dengue haemorrhagic epidemics and the information could be used to implement appropriate prevention and control measures. Virological surveillance was essential in order to detected this new serotype.

Complete genetic characterization of a Brazilian dengue virus type 3 strain isolated from a fatal outcome

We have determined the complete nucleotide and the deduced amino acid sequences of Brazilian dengue virus type 3 (DENV-3) from a dengue case with fatal outcome, which occurred during an epidemic in the state of Rio de Janeiro, Brazil, in 2002. This constitutes the first complete genetic characterization of a Brazilian DENV-3 strain since its introduction into the country in 2001. DENV-3 was responsible for the most severe dengue epidemic in the state, based on the highest number of reported cases and on the severity of clinical manifestations and deaths reported.

Dengue virus type 3 in Brazil: a phylogenetic perspective

Circulation of a new dengue virus (DENV)-3 genotype was recently described in Brazil and Colombia, but the precise classification of this genotype has been controversial. Here we perform phylogenetic and nucleotide-distance analyses of the envelope gene, which support the subdivision of DENV-3 strains into five distinct genotypes (GI to GV) and confirm the classification of the new South American genotype as GV. The extremely low genetic distances between Brazilian GV strains and the prototype Philippines/L11423 GV strain isolated in 1956 raise important questions regarding the origin of GV in South America.

Construction and characterisation of a complete reverse genetics system of dengue virus type 3

Dengue virulence and fitness are important factors that determine disease outcome. However, dengue virus (DENV) molecular biology and pathogenesis are not completely elucidated. New insights on those mechanisms have been facilitated by the development of reverse genetic systems in the past decades. Unfortunately, instability of flavivirus genomes cloned in Escherichia coli has been a major problem in these systems. Here, we describe the development of a complete reverse genetics system, based on the construction of an infectious clone and replicon for a low passage DENV-3 genotype III of a clinical isolate. Both constructs were assembled into a newly designed yeast- E. coli shuttle vector by homologous recombination technique and propagated in yeast to prevent any possible genome instability in E. coli . RNA transcripts derived from the infectious clone are infectious upon transfection into BHK-21 cells even after repeated passages of the plasmid in yeast. Transcript-derived DENV-3 exhibited growth kinetics, focus formation size comparable to original DENV-3 in mosquito C6/36 cell culture. In vitro characterisation of DENV-3 replicon confirmed its identity and ability to replicate transiently in BHK-21 cells. The reverse genetics system reported here is a valuable tool that will facilitate further molecular studies in DENV replication, virus attenuation and pathogenesis.

Dengue viruses activity in Piauí, Brazil

The present paper reports a laboratory investigation performed between the years of 2000 and 2002 to stydy a virological surveillance program introduced in the state of Piauí to support an epidemiological survey of the disease. Dengue virus type 3 (DENV-3) existence in the state was detected in May 2002 when a high number of dengue cases due to DENV-1 and DENV-2 were reported. An assessment on the population knowledge about the disease and its transmission showed that almost 50% of the population were still unaware of the epidemiological features of dengue.

Brazilian dengue virus type 1 replication in mosquito cell cultures

The development of dengue viruses type 1 obtained from accute human sera and inoculated into mosquito cell cultures, was observed by standard transmission electron microscopy and cytochemical staining. It follows the trans-type mechanism already estabilished of other dengue types. Directed passage of single virus particles across the cell membrane seems to be a pathway of entry and exit in dengue-1 infected cells. The nature of numerous electron translucent vesicles and tubules, produced simmultaneously during virus replication inside the rough endoplasmic reticulum, was analyzed by cytochemical tests. The largest amount of virus particles was produced inside cell syncytia.

Comparative immunological recognition of proteins from New Guinea "C" dengue virus type 2 prototype and from a dengue virus type 2 strain isolated in the State of Rio de Janeiro, Brazil

The protein profiles of the New Guinea "C" dengue virus type 2 (DENV-2)prototype and those of a Brazilian DENV-2 isolated in the State of Rio de Janeiro in 1995 were compared. SDS-PAGE analysis showed that the virus from Rio de Janeiro expresses NS5 (93.0 kDa), NS3 (66.8 kDa) E (62.4 kDa) and NS1 (41.2 kDa) proteins differently from the New Guinea "C" virus. The immunoblot revealed specificity and antigenicity for the NS3 protein from DENV-2 Rio de Janeiro mainly in primary infections, convalescent cases, and in secondary infections in both cases and only antigenicity for E and NS1 proteins for both viruses in primary and secondary infections.

Concurrent infection with dengue virus type-2 and DENV-3 in a patient from Ceará, Brazil

Dengue outbreaks have occurred in several regions in Brazil and cocirculating dengue virus type 1 (DENV-1), DENV-2, and DENV-3 have been frequently observed. Dual infection by DENV-2 and DENV-3 was identified by type-specific indirect immunofluorescence assay and confirmed by reverse transcription polymerase chain reaction in a patient in Ceará with a mild disease. This is the first documented case of simultaneous infection with DENV-2 and DENV-3 in Brazil. Sequencing confirmed DENV-2 and DENV-3 (South-East/American) genotype III and (SriLanka/India), genotype III respectively.

Molecular epidemiology of type 1 and 2 dengue viruses in Brazil from 1988 to 2001

Dengue is a mosquito-borne viral infection that in recent decades has become a major international public health concern. Epidemic dengue fever reemerged in Brazil in 1981. Since 1990 more than one dengue virus serotype has been circulating in this tropical country and increasing rates of dengue hemorrhagic fever and dengue shock syndrome have been detected every year. Some evidence supports the association between the introduction of a new serotype and/or genotype in a region and the appearance of dengue hemorrhagic fever. In order to study the evolutionary relationships and possible detection of the introduction of new dengue virus genotypes in Brazil in the last years, we analyzed partial nucleotide sequences of 52 Brazilian samples of both dengue type 1 and dengue type 2 isolated from 1988 to 2001 from highly endemic regions. A 240-nucleotide-long sequence from the envelope/nonstructural protein 1 gene junction was used for phylogenetic analysis. After comparing the nucleotide sequences originally obtained in this study to those previously studied by others, and analyzing the phylogenetic trees, we conclude that, after the initial introduction of the currently circulating dengue-1 and dengue-2 genotypes in Brazil, there has been no evidence of introduction of new genotypes since 1988. The increasing number of dengue hemorrhagic fever cases seen in Brazil in the last years is probably associated with secondary infections or with the introduction of new serotypes but not with the introduction of new genotypes.

Molecular typing of dengue virus type 2 in Brazil

Strain typing is a critical tool for molecular epidemiological analysis and can provide important information about the spread of dengue viruses. Here, we performed a molecular characterization of DEN-2 viruses isolated in Brazil during 1990-2000 from geographically and temporally distinct areas in order to investigate the genetic distribution of this serotype circulating in the country. Restriction site-specific polymerase chain reaction (RSS)-PCR presented the same pattern for all 52 Brazilian samples, showing the circulation of just one DEN-2 variant. Phylogenetic analysis using progressive pairwise alignments from 240-nucleotide sequences of the E/NS1 junction in 15 isolates showed that they belong to genotype III (Jamaica genotype).

Molecular analysis of the dengue virus type 1 and 2 in Brazil based on sequences of the genomic envelope-nonstructural protein 1 junction region

The genomic sequences of the Envelope-Non-Structural protein 1 junction region (E/NS1) of 84 DEN-1 and 22 DEN-2 isolates from Brazil were determined. Most of these strains were isolated in the period from 1995 to 2001 in endemic and regions of recent dengue transmission in São Paulo State. Sequence data for DEN-1 and DEN-2 utilized in phylogenetic and split decomposition analyses also include sequences deposited in GenBank from different regions of Brazil and of the world. Phylogenetic analyses were done using both maximum likelihood and Bayesian approaches. Results for both DEN-1 and DEN-2 data are ambiguous, and support for most tree bipartitions are generally poor, suggesting that E/NS1 region does not contain enough information for recovering phylogenetic relationships among DEN-1 and DEN-2 sequences used in this study. The network graph generated in the split decomposition analysis of DEN-1 does not show evidence of grouping sequences according to country, region and clades. While the network for DEN-2 also shows ambiguities among DEN-2 sequences, it suggests that Brazilian sequences may belong to distinct subtypes of genotype III.