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Abstract The number of dengue cases has increased dramatically in recent years. In Latin America, the number of cases and deaths in 2023 was the highest ever recorded. We report on a patient who had been infected with dengue virus during his stay in Costa Rica in September 2023, and developed the disease after returning to Japan. Plasma obtained from the patient was used for diagnosis and dengue virus serotyping by real-time PCR. The nucleotide sequence of the envelope region of dengue virus was then determined by the direct sequencing method, and this sequence was used for phylogenetic analyses. The patient was found to be infected with dengue virus type 3 genotype III. The sequence from the present case was more homologous with sequences registered in Florida, USA, associated with travel to Cuba in 2022 than with sequences registered in Costa Rica 10 years ago. The Pan American Health Organization reported that only dengue virus type 1 and 2 cases were reported in Costa Rica in 2019–2021, whereas dengue virus type 3 and 4 cases started being reported in 2022. In 2023, the reported numbers of cases with dengue virus types 3 and 4 exceeded those of dengue virus types 1 and 2. In addition, regional differences in endemic strains have been observed in Costa Rica. Our findings suggest that the dengue virus type 3 that infected the patient was more likely an influx of a strain that had been circulating in Caribbean countries such as Cuba in recent years, rather than a re-emergence of an indigenous virus in Costa Rica. The serotypes of dengue virus prevalent in Costa Rica have been changing since 2022. All four serotypes were prevalent in 2023, with a particularly sharp increase in the number of cases of dengue virus types 3 and 4. Future monitoring and surveillance are essential because changes in endemic serotypes can cause antibody-dependent enhancement, which can lead to severe dengue disease presentations.

The number of dengue cases has increased dramatically in recent years. In Latin America, the number of cases and deaths in 2023 was the highest ever recorded. We report on a patient who had been infected with dengue virus during his stay in Costa Rica in September 2023, and developed the disease after returning to Japan. Plasma obtained from the patient was used for diagnosis and dengue virus serotyping by real-time PCR. The nucleotide sequence of the envelope region of dengue virus was then determined by the direct sequencing method, and this sequence was used for phylogenetic analyses. The patient was found to be infected with dengue virus type 3 genotype III. The sequence from the present case was more homologous with sequences registered in Florida, USA, associated with travel to Cuba in 2022 than with sequences registered in Costa Rica 10 years ago. The Pan American Health Organization reported that only dengue virus type 1 and 2 cases were reported in Costa Rica in 2019–2021, whereas dengue virus type 3 and 4 cases started being reported in 2022. In 2023, the reported numbers of cases with dengue virus types 3 and 4 exceeded those of dengue virus types 1 and 2. In addition, regional differences in endemic strains have been observed in Costa Rica. Our findings suggest that the dengue virus type 3 that infected the patient was more likely an influx of a strain that had been circulating in Caribbean countries such as Cuba in recent years, rather than a re-emergence of an indigenous virus in Costa Rica. The serotypes of dengue virus prevalent in Costa Rica have been changing since 2022. All four serotypes were prevalent in 2023, with a particularly sharp increase in the number of cases of dengue virus types 3 and 4. Future monitoring and surveillance are essential because changes in endemic serotypes can cause antibody-dependent enhancement, which can lead to severe dengue disease presentations. [ABSTRACT FROM AUTHOR]

Insect-specific flaviviruses (ISFs) have been isolated from a range of mosquito species from different parts of the world. These viruses replicate efficiently in mosquitoes but do not appear to replicate in vertebrates. There is increasing evidence that ISFs persist in nature through vertical transmission, and that they interfere with the replication and transmission of pathogenic flaviviruses in the mosquito host. A novel ISF species, Parramatta River virus (PaRV), was previously shown to occur at high rates in Aedes (Ae.) vigilax mosquitoes collected from Sydney, Australia. We investigated whether vertical transmission was the mechanism of viral persistence in Ae. vigilax populations and whether PaRV affected replication of the pathogenic flaviviruses, West Nile virus (WNV), and dengue virus type 3 (DENV-3) in cultured mosquito cells. Progeny reared from eggs obtained from field-collected infected females had infection rates as high as 142 and 85 per 1000 for females and males, respectively. In vitro experiments showed that replication of both WNV and DENV-3 was significantly suppressed in Aedes albopictus (C6/36) cells persistently infected with PaRV. Our studies with PaRV support the findings of previous investigations that ISFs persist in nature through vertical transmission and that ISFs can suppress the replication of pathogenic flaviviruses in coinfected mosquito cells. [ABSTRACT FROM AUTHOR]

In the past few decades, dengue has spread rapidly and is an emerging disease in China. An unexpected dengue outbreak occurred in Xishuangbanna, Yunnan, China, resulting in 1331 patients in 2013. In order to obtain the complete genome information and perform mutation and evolutionary analysis of causative agent related to this largest outbreak of dengue fever. The viruses were isolated by cell culture and evaluated by genome sequence analysis. Phylogenetic trees were then constructed by Neighbor-Joining methods (MEGA6.0), followed by analysis of nucleotide mutation and amino acid substitution. The analysis of the diversity of secondary structure for E and NS1 protein were also performed. Then selection pressures acting on the coding sequences were estimated by PAML software. The complete genome sequences of two isolated strains (YNSW1, YNSW2) were 10,710 and 10,702 nucleotides in length, respectively. Phylogenetic analysis revealed both strain were classified as genotype II of DENV-3. The results indicated that both isolated strains of Xishuangbanna in 2013 and Laos 2013 stains (KF816161.1, KF816158.1, LC147061.1, LC147059.1, KF816162.1) were most similar to Bangladesh (AY496873.2) in 2002. After comparing with the DENV-3SS (H87) 62 amino acid substitutions were identified in translated regions, and 38 amino acid substitutions were identified in translated regions compared with DENV-3 genotype II stains Bangladesh (AY496873.2). 27(YNSW1) or 28(YNSW2) single nucleotide changes were observed in structural protein sequences with 7(YNSW1) or 8(YNSW2) non-synonymous mutations compared with AY496873.2. Of them, 4 non-synonymous mutations were identified in E protein sequences with (2 in the β-sheet, 2 in the coil). Meanwhile, 117(YNSW1) or 115 (YNSW2) single nucleotide changes were observed in non-structural protein sequences with 31(YNSW1) or 30 (YNSW2) non-synonymous mutations. Particularly, 14 single nucleotide changes were observed in NS1 sequences with 4/14 non-synonymous substitutions (4 in the coil). Selection pressure analysis revealed no positive selection in the amino acid sites of the genes encoding for structural and non-structural proteins. This study may help understand the intrinsic geographical relatedness of dengue virus 3 and contributes further to research on their infectivity, pathogenicity and vaccine development. [ABSTRACT FROM AUTHOR]

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.

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.

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.

Highlights: [•] We documented the first evidence of recombination between DENV-3 viruses. [•] Two viruses represent recombination between viruses from different genotypes. [•] We identified multiple breakpoints sites in the recombinant viruses. [•] Multiple strand switching may be linked with the genomic mosaicism. [ABSTRACT FROM AUTHOR]

Background: Investigations were conducted by the authors to explore an outbreak of viral hemorrhagic fever (VHF) reported in 2010 from Al-Mukalla city, the capital of Hadramout in Yemen. Methods: From 15-17 June 2010, the outbreak investigation period, specimens were obtained within 7 days after onset of illness of 18 acutely ill patients hospitalized with VHF and 15 household asymptomatic contacts of 6 acute cases. Additionally, 189 stored sera taken from acutely ill patients with suspected VHF hospitalized in the preceding 12 months were obtained from the Ministry of Health of Yemen. Thus, a total of 222 human specimens were collected; 207 specimens from acute cases and 15 specimens from contacts. All samples were tested with RT-PCR for dengue (DENV), Alkhumra (ALKV), Rift Valley Fever (RVFV), Yellow Fever (YFV), and Chikungunya (CHIKV) viruses. Samples were also tested for DENV IgM, IgG, and NS1-antigen. Medical records of patients were reviewed and demographic, clinical, and laboratory data was collected. Results: Of 207 patients tested, 181 (87.4%) patients were confirmed to have acute dengue with positive dengue NS1-antigen (97 patients, 46.9%) and/or IgM (163 patients, 78.7%). Of the 181 patients with confirmed dengue, 100 (55.2%) patients were IgG-positive. DENV RNA was detected in 2 (1%) patients with acute symptoms; both samples were molecularly typed as DENV type 3. No other VHF viruses were detected. For the 15 contacts tested, RT-PCR tests for the five viruses were negative, one contact was dengue IgM positive, and another one was dengue IgG positive. Of the 181 confirmed dengue patients, 120 (66.3%) patients were males and the median age was 24 years. The most common manifestations included fever (100%), headache (94.5%), backache (93.4%), malaise (88.4%), arthralgia (85.1%), myalgia (82.3%), bone pain (77.9%), and leukopenia (76.2%). Two (1.1%) patients died. Conclusions: DENV-3 was confirmed to be the cause of an outbreak of VHF in Al-Mukalla. It is important to use both IgM and NS1-antigen tests to confirm acute dengue particularly under the adverse field conditions, where proper storage and transportation of specimens are missing, which substantially reduce the sensitivity of the RT-PCR for detecting DENV RNA. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Dengue is emerging as the most important mosquito borne viral disease in the world. In mainland China, sporadic and large outbreaks of dengue illness caused by the four serotypes of dengue virus (DENV-1 to DENV-4) have been well documented. Guangdong province is the major affected area in China, and DENV-1 has dominantly circulated in Guangdong for a long time. In this study, a family cluster of DENV-3 infection in Guangzhou was described. Three cases were diagnosed as dengue fever based on clinical manifestation, serological and RT-PCR assays. Two DENV-3 strains were isolated in C6/36 cells and the complete genome sequences were determined. Phylogenetic analysis revealed that the new DENV-3 isolates from the family cluster were grouped within genotype III. Considering the fact that several DENV-3 strains within genotype V were also identified in Guangzhou in 2009, at least two genotypes of DENV-3 co-circulated in Guangzhou. Careful investigation and virological analysis should be warranted in the future. [ABSTRACT FROM AUTHOR]

Dengue virus type 3 (DENV-3) re-appeared in Colombia in 2001 after 23 years of apparent absence, in the state of Santander in the North-eastern region near to Venezuelan border. In 2002, the virus was isolated in the state of Valle del Cauca in the South-east region near to Ecuadorian/Peruvian border, and in the state of Antioquia in the North-east region near to Panama border. To gain insight into the molecular epidemiology of DENV-3 in Colombia, we sequenced the complete E gene of 21 isolates sampled in the period 2001–2007. Phylogenetic analyses revealed that Colombian strains seem to have been introduced from Venezuela, Ecuador and Peru, but not from Brazil, Argentina, Paraguay or Central America countries. This study also confirms previous report showing that Colombian isolates is closely related to DENV-3 genotype III. [ABSTRACT FROM AUTHOR]

Abstract: In this study, we revisited the phylogeography of the three of major DENV-3 genotypes and estimated its rate of evolution, based on the analysis of the envelope (E) gene of 200 strains isolated from 31 different countries around the world over a time period of 50 years (1956–2006). Our phylogenetic analysis revealed a geographical subdivision of DENV-3 population in several country-specific clades. Migration patterns of the main DENV-3 genotypes showed that genotype I was mainly circumspect to the maritime portion of Southeast-Asia and South Pacific, genotype II stayed within continental areas in South-East Asia, while genotype III spread across Asia, East Africa and into the Americas. No evidence for rampant co-circulation of distinct genotypes in a single locality was found, suggesting that some factors, other than geographic proximity, may limit the continual dispersion and reintroduction of new DENV-3 variants. Estimates of the evolutionary rate revealed no significant differences among major DENV-3 genotypes. The mean evolutionary rate of DENV-3 in areas with long-term endemic transmissions (i.e., Indonesia and Thailand) was similar to that observed in the Americas, which have been experiencing a more recent dengue spread. We estimated the origin of DENV-3 virus around 1890, and the emergence of current diversity of main DENV-3 genotypes between the middle 1960s and the middle 1970s, coinciding with human population growth, urbanization, and massive human movement, and with the description of the first cases of DENV-3 hemorrhagic fever in Asia. [Copyright &y& Elsevier]

Abstract Dengue is emerging as the most important mosquito borne viral disease in the world. In mainland China, sporadic and large outbreaks of dengue illness caused by the four serotypes of dengue virus (DENV-1 to DENV-4) have been well documented. Guangdong province is the major affected area in China, and DENV-1 has dominantly circulated in Guangdong for a long time. In this study, a family cluster of DENV-3 infection in Guangzhou was described. Three cases were diagnosed as dengue fever based on clinical manifestation, serological and RT-PCR assays. Two DENV-3 strains were isolated in C6/36 cells and the complete genome sequences were determined. Phylogenetic analysis revealed that the new DENV-3 isolates from the family cluster were grouped within genotype III. Considering the fact that several DENV-3 strains within genotype V were also identified in Guangzhou in 2009, at least two genotypes of DENV-3 co-circulated in Guangzhou. Careful investigation and virological analysis should be warranted in the future.

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.

Summary: We examined levels of dengue virus type 3 (DENV-3) RNA in association with the type of infection (primary or secondary) in 42 patients with fatal and non-fatal outcomes in Rio de Janeiro, Brazil, 2002. Subjects with fatal outcomes had mean virus titers significantly higher than those who survived (12.5 vs. 7.9 log10 RNA copies/ml). Because primary infections were confirmed among the fatal cases (52.1%), antibody-dependent enhancement alone did not explain all the cases of severe disease in this study population. These findings suggest that high levels of DENV-3 may have contributed to the severe form of dengue in Rio de Janeiro, 2002. [Copyright &y& Elsevier]

Summary: Background: After an absence of 29 years, dengue virus type 3 (DENV-3) re-emerged in Guangzhou in 2009 and again in 2010. However, the geographical route by which the virus entered the city, and how it has changed genetically, remain unclear. Therefore, we carried out a comprehensive investigation into the molecular characteristics of the DENV-3 involved. Methods: The envelope (E) genes of viruses isolated from dengue patients during the 2009–2010 epidemics were sequenced and compared with previously published E gene sequences of global representative DENV-3 strains available in GenBank, including isolates circulating in other provinces of China. Results: A total of 13 isolates (seven from 2009 and six from 2010) were obtained from human serum samples. Phylogenetic analysis revealed that the isolates were grouped into three genotypes (I, III, and V) and then two clades within genotype III (genotype I from Indonesia, genotype III clade A from Côte d’Ivoire, genotype III clade B from Tanzania, and genotype V from Philippines). In addition, there were 1.3–9.0% and 0.5–3.9% differences in the nucleic and deduced amino acid sequences between the 2009 and 2010 strains, respectively. Conclusions: The DENV-3 viruses from the period 2009–2010 were not from the continuous spread of an epidemic strain or the re-emergence of the 2009 strains in the 2-year period. The introduction of different DENV-3 genotypes following more than one geographical route was an important contributing factor to the 2009–2010 dengue epidemics in Guangzhou. [ABSTRACT FROM AUTHOR]

Summary: Objectives: Recognizing the uniqueness of secondary dengue virus (DENV)-1/3 dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) cases at an interval of 24 years, we sought to estimate DENV infections as well as the ratios between mild disease and DHF/DSS by DENV infection sequence in Playa District (Havana, Cuba) during the 2001–2002 outbreak of dengue virus type 3 (DENV-3). Methods: A retrospective seroepidemiological study was conducted in 2003 in Playa District. Blood samples were collected from a 1% random sample of residents and were studied for the prevalence of dengue neutralizing antibodies. Results: DENV-3 was found to have infected 7.2% (95% confidence interval (95% CI) 6.0–8.4%) of susceptible individuals (the entire cohort), the majority of whom experienced silent infections. Virtually every individual who had a secondary infection in the sequence DENV-1 then DENV-3 became ill, with a ratio of severe to mild cases of 1:35 (95% CI 1:67–1:23). Secondary infections in the sequence DENV-2/3 were less pathogenic than DENV-1/3. Mild disease accompanying secondary DENV2/3 occurred at a ratio of 1:4.49 infections (95% CI 1:5.77–1:3.42) secondary infections. Conclusions: The results obtained highlight the role of the infecting serotype and also the sequence of the viral infection in the clinical outcome of a dengue infection. [Copyright &y& Elsevier]

After an 18-year absence, dengue virus serotype 3 reemerged in the South Pacific Islands in 2013. Outbreaks in western (Solomon Islands) and eastern (French Polynesia) regions were caused by different genotypes. This finding suggested that immunity against dengue virus serotype, rather than virus genotype, was the principal determinant of reemergence.