Genetic variation in the 3’ untranslated region of dengue virus serotype 3 strains isolated from mosquitoes and humans in Brazil

SUMMARY: BACKGROUND: Dengue, a mosquito-borne viral infection caused by one of the four dengue virus (DENV) serotypes (DENV-1 to 4), replicate alternately on the mosquito vector and human host and are responsible for infections throughout tropical and subtropical regions of the world. In Brazil, the disease has become a major public health problem and the introduction of DENV-3 in 2000 in Rio de Janeiro (RJ) was associated with severe dengue epidemics. The potential emergence of strains associated with severe disease highlights the need for the surveillance of DENV in human host and vectors. METHODS: Aiming to contribute for DENV phylogenetic and vector-virus-human host studies, we sequenced the entire genome of one DENV-3 isolated from naturally infected Aedes aegypti from RJ in 2001 and characterized the 3’ UTR from strains isolated from mosquitoes and humans. Mosquitoes were pooled and submitted to virus isolation in Ae. albopictus C6/36 cells and the infecting serotype was identified by immunofluorescence using type-specific monoclonal antibody. Sequence analysis was performed using BioEdit software, the multiple alignments were performed using CLUSTAL W and the phylogenetic analysis by MEGA 5, using the Neighbor-joining method. Secondary structure prediction was performed by using the MFOLD program. RESULTS: Exclusive substitutions and a substitution leading to a stop codon on the NS5 gene were observed in the DENV-3 isolated from a naturally infected Ae. aegypti and fully sequenced. As an 8- nucleotides deletion was observed within the 11- nucleotides (nts) insertion on the variable region (VR) from the 3′UTR in this isolate, we further sequenced other DENV-3 from both mosquitoes and humans. The majority of DENV-3 from RJ analyzed were characterized by the 11-nts insertion in the VR of the 3′UTR, despite the observation of strains carrying the 8-nts deletion. The latter presented similar secondary structures, however not all strains presenting the 11-nts insertion were similar in the predicted secondary structure. CONCLUSIONS: The phylogeny based on the analysis of the complete genome and 3′UTR characterized the DENV-3 isolated from both vector and human host as belonging to Genotype III (GIII), despite the differences observed on the 3’ UTR. Further studies are needed to address the role of those mutations in the transmission of the different viral populations and vector competence

Genetic variation in the 3’ untranslated region of dengue virus serotype 3 strains isolated from mosquitoes and humans in Brazil

Summary Background Dengue, a mosquito-borne viral infection caused by one of the four dengue virus (DENV) serotypes (DENV-1 to 4), replicate alternately on the mosquito vector and human host and are responsible for infections throughout tropical and subtropical regions of the world. In Brazil, the disease has become a major public health problem and the introduction of DENV-3 in 2000 in Rio de Janeiro (RJ) was associated with severe dengue epidemics. The potential emergence of strains associated with severe disease highlights the need for the surveillance of DENV in human host and vectors. Methods Aiming to contribute for DENV phylogenetic and vector-virus-human host studies, we sequenced the entire genome of one DENV-3 isolated from naturally infected Aedes aegypti from RJ in 2001 and characterized the 3’ UTR from strains isolated from mosquitoes and humans. Mosquitoes were pooled and submitted to virus isolation in Ae. albopictus C6/36 cells and the infecting serotype was identified by immunofluorescence using type-specific monoclonal antibody. Sequence analysis was performed using BioEdit software, the multiple alignments were performed using CLUSTAL W and the phylogenetic analysis by MEGA 5, using the Neighbor-joining method. Secondary structure prediction was performed by using the MFOLD program. Results Exclusive substitutions and a substitution leading to a stop codon on the NS5 gene were observed in the DENV-3 isolated from a naturally infected Ae. aegypti and fully sequenced. As an 8- nucleotides deletion was observed within the 11- nucleotides (nts) insertion on the variable region (VR) from the 3′UTR in this isolate, we further sequenced other DENV-3 from both mosquitoes and humans. The majority of DENV-3 from RJ analyzed were characterized by the 11-nts insertion in the VR of the 3′UTR, despite the observation of strains carrying the 8-nts deletion. The latter presented similar secondary structures, however not all strains presenting the 11-nts insertion were similar in the predicted secondary structure. Conclusions The phylogeny based on the analysis of the complete genome and 3′UTR characterized the DENV-3 isolated from both vector and human host as belonging to Genotype III (GIII), despite the differences observed on the 3’ UTR. Further studies are needed to address the role of those mutations in the transmission of the different viral populations and vector competence.</p

Re-introduction of dengue virus serotype 2 in the state of Rio de Janeiro after almost a decade of epidemiological silence.

The Asian/American genotype of dengue virus serotype 2 (DENV-2) has been introduced in Brazil through the state of Rio de Janeiro around 1990, and since then it has been spreading and evolving, leading to several waves of dengue epidemics throughout the country that cause a major public health problem. Of particular interest has been the epidemic of 2008, whose highest impact was evidenced in the state of Rio de Janeiro, with a higher number of severe cases and mortality rate, compared to previous outbreaks. Interestingly, no circulation of DENV-2 was witnessed in this region during the preceding 9-year period. By early 2010, phylogenetic analysis of the 2008 epidemic strain revealed that the outbreak was caused by a new viral lineage of the Asian/American genotype, which was pointed as responsible for the outbreak severity as well. The same scenario is repeating in 2019 in this state; however, only a few cases have been detected yet. To provide information that helps to the understanding of DENV-2 dynamics in the state of Rio de Janeiro, and thereafter contribute to public health control and prevention actions, we employed phylogenetic studies combined with temporal and dynamics geographical features to determine the origin of the current viral strain. To this effect, we analyzed a region of 1626 nucleotides entailing the Envelope/NS1 viral genes. Our study reveals that the current strain belongs to the same lineage that caused the 2008 outbreak, however, it is phylogenetically distant from any Brazilian strain identified so far. Indeed, it seemed to be originated in Puerto Rico around 2002 and has been introduced into the state in late 2018. Taking into account that no DENV-2 case was reported over the last decade in the state (representing a whole susceptible children generation), and the fact that a new viral strain may be causing current dengue infections, these results will be influential in strengthening dengue surveillance and disease control, mitigating the potential epidemiological consequences of virus spread

Dengue 4 in Ceará, Brazil: characterisation of epidemiological and laboratorial aspects and causes of death during the first epidemic in the state

BACKGROUND The first dengue cases in Brazil with laboratory confirmation occurred in the northern region of the country, with the isolation of two serotypes, dengue virus 1 (DENV-1) and DENV-4. In Ceará, the introduction of DENV-4 was reported during a DENV-1 epidemic in 2011, with only two isolations. OBJECTIVES The aim of this study was to characterise the first DENV-4 epidemic in the state of Ceará, Brazil. METHODS The study population was composed of patients with suspected dengue that were reported to health care units from January to December 2012. The laboratory confirmation of infection was made by viral isolation, reverse transcription polymerase chain reaction (RT-PCR), AgNS1, immunohistochemistry and IgM enzyme-linked immunosorbent assay (ELISA). MAIN CONCLUSIONS In the study year, 72,211 suspected dengue cases were reported and 51,865 of these cases (71.8%) were confirmed to be positive. Co-circulation of three serotypes, DENV-1, DENV-3 and DENV-4, was detected with a predominance of DENV-4 (95.3%). Most cases were not severe, but there were 44 fatal outcomes. DENV-4 Genotype II was identified for the first time in Ceará

Dengue type 4 in Rio de Janeiro, Brazil: case characterization following its introduction in an endemic region

Abstract Background Due to the populations’ susceptibility, DENV-4 introduction in 2010 led to the occurrence of explosive epidemics in the following years in Brazil. In 2011, DENV-4 was identified in Rio de Janeiro (RJ) and it was prevalent in 2012 and 2013. Here, we aimed to characterize clinical, epidemiological and laboratorial aspects of DENV-4 cases after this serotype introduction in an endemic scenario. Methods Dengue suspected cases (n = 3727) were received and analyzed from January 2011 to December 2013, during outbreaks occurred in RJ, Brazil. Samples were submitted to virological, serological and molecular methods for case confirmation. DENV-4 cases (n = 705) were characterized according to the type of infection, disease severity and, viremia levels and NS1 antigenemia were accessed. Representative strains were partial sequenced for genotyping. Results DENV-4 was identified in 44.2% (705/1593) of dengue positive cases, virus isolated in 48.7% of the cases. Anti-DENV IgM was detected in 39.4% of the cases, however an increased detection was observed in cases with ≥4 days of symptoms (57.0%). NS1 antigen was identified in 41.5% of DENV-4 cases however, after immune complexes dissociation, the detection significantly increased (87.6%). Females were more affected than males, so did children aged 11–15 years old. Primary cases were more frequently observed than secondary ones and most of them were classified as dengue. No differences on NS1 antigenemia and viraemia within the groups were observed. Despite the higher frequency of severe disease on individuals >65 years old, no differences were observed among the groups and type of infection. However, DENV-4 fatal cases were more frequent on secondary infections (57.1%). DENV-4 Genotype II was identified with a probable origin from Venezuela and Colombia. Conclusions It has been shown that laboratorial diagnosis is still a reliable tool for the disease surveillance, detecting and confirming emerging epidemics. Despite the occurrence of secondary infections, most DENV-4 cases presented a mild disease. As RJ is endemic for dengue, high rates of secondary infections would be expected. Despite the existence of two genotypes, only Genotype II was identified in our study

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

<div><p>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 (<i>n</i> = 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.</p> </div

DENV-1 Genotype V in Brazil: Spatiotemporal dispersion pattern reveals continuous co-circulation of distinct lineages until 2016

Submitted by Sandra Infurna ([email protected]) on 2019-02-08T15:38:54Z No. of bitstreams: 1 fernandaB_nogueira_etal_IOC_2018.pdf: 2567629 bytes, checksum: 2b64c7ba0484dc18ff101c9638713946 (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2019-02-08T15:54:49Z (GMT) No. of bitstreams: 1 fernandaB_nogueira_etal_IOC_2018.pdf: 2567629 bytes, checksum: 2b64c7ba0484dc18ff101c9638713946 (MD5)Made available in DSpace on 2019-02-08T15:54:49Z (GMT). No. of bitstreams: 1 fernandaB_nogueira_etal_IOC_2018.pdf: 2567629 bytes, checksum: 2b64c7ba0484dc18ff101c9638713946 (MD5) Previous issue date: 2018Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Imunologia Viral. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Imunologia Viral. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Imunologia Viral. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Flavivírus. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Flavivírus. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Flavivírus. Rio de Janeiro, RJ, Brasil.Laboratório Central de Saúde Pública do Ceará. Fortaleza, CE, Brasil.Laboratório Central de Saúde Pública do Rio de Janeiro. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Flavivírus. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Flavivírus. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Imunologia Viral. Rio de Janeiro, RJ, Brasil.In Brazil, DENV-1 introduced in the 80's, remained the prevalent serotype from 2012 to 2016. After its re-emergence in the country in 2009, the co-circulation of different viral lineages was identified, however, its transmission dynamics afterwards, was not fully characterized. In this study, we performed the continuous molecular surveillance after the reemergence period (2012 to 2016), covering the 30 years of circulation of DENV-1 in Brazil. Phylogenetic analysis allowed confirmation of the continued presence of genotype V, as well as three distinct co-circulating lineages. The molecular characterization of the E gene presented two new amino acid substitutions previously unidentified in the country. Phylogeographic analysis has shown that a large flow of migrations has occurred between Brazil and Argentina in the last 10 years

Maximum likelihood phylogeny based on the C/prM/M/E genes of 25 Brazilian DENV-2, 1991–2010.

<p>Black circles represent DENV-2 sequences generated in this study. Strains representative from the four genotypes available in Genbank (<a href="http://www.ncbi.nlm.nih.gov" target="_blank">www.ncbi.nlm.nih.gov</a>) were used for the comparison, DENV-1, DENV-3 and DENV-4 strains were used as outgroup to root the trees. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. DENV strains used were named as follows: Country/strain number/state/year. RJ: Rio de Janeiro, ES: Espirito Santo, CE: Ceará, BA: Bahia, RS: Rio Grande do Sul, RN: Rio Grande do Norte.</p

Strains representative of the different DENV-2 genotypes and strains used as outgroup for comparison purposes.

<p>Strains representative of the different DENV-2 genotypes and strains used as outgroup for comparison purposes.</p

Primers used for amplification of the partial and complete genes (coding region) from Brazilian DENV-2.

<p>Primers used for amplification of the partial and complete genes (coding region) from Brazilian DENV-2.</p