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.     

Complete Genome Sequence of Dengue Virus Serotype 2 Cosmopolitan Genotype Strain in Guangdong,China

Here we report the complete genome sequence of a dengue virus serotype 2 (DENV-2) strain, GZ40, isolated in Guangdong, China, in 2010. A phylogenetic analysis classified GZ40 into the Cosmopolitan genotype, while previous Chinese DENV-2 isolates belong to the Asian I genotype. The reemergence of the Cosmopolitan genotype of DENV-2 in China deserves further investigation.     

Circulation of Different Lineages of Dengue Virus 2, Genotype American/Asian in Brazil: Dynamics and Molecular and Phylogenetic Characterization

The American/Asian genotype of Dengue virus type 2 (DENV-2) was introduced into the Americas in the 80′s. Although there is no data showing when this genotype was first introduced into Brazil, it was first detected in Brazil in 1990. After which the virus spread throughout the country and major epidemics occurred in 1998, 2007/08 and 2010. In this study we sequenced 12 DENV-2 genomes obtained from serum samples of patients with dengue fever residing in São José do Rio Preto, São Paulo (SJRP/SP), Brazil, in 2008. The whole open reading frame or envelope sequences were used to perform phylogenetic, phylogeographic and evolutionary analyses. Isolates from SJRP/SP were grouped within one lineage (BR3) close to isolates from Rio de Janeiro, Brazil. Isolates from SJRP were probably introduced there at least in 2007, prior to its detection in the 2008 outbreak. DENV-2 circulation in Brazil is characterized by the introduction, displacement and circulation of three well-defined lineages in different times, most probably from the Caribbean. Thirty-seven unique amino acid substitutions were observed among the lineages, including seven amino acid differences in domains I to III of the envelope protein. Moreover, we dated here, for the first time, the introduction of American/Asian genotype into Brazil (lineage BR1) to 1988/89, followed by the introduction of lineages BR2 (1998–2000) and BR3 (2003–05). Our results show a delay between the introduction and detection of DENV-2 lineages in Brazil, reinforcing the importance and need for surveillance programs to detect and trace the evolution of these viruses. Additionally, Brazilian DENV-2 differed in genetic diversity, date of introduction and geographic origin and distribution in Brazil, and these are important factors for the evolution, dynamics and control of dengue.     

Circulation of different lineages of dengue virus type 2 in Central America, their evolutionary time-scale and selection pressure analysis

Dengue is caused by any of the four serotypes of dengue virus (DENV-1 to 4). Each serotype is genetically distant from the others, and each has been subdivided into different genotypes based on phylogenetic analysis. The study of dengue evolution in endemic regions is important since the diagnosis is often made by nucleic acid amplification tests, which depends upon recognition of the viral genome target, and natural occurring mutations can affect the performance of these assays. Here we report for the first time a detailed study of the phylogenetic relationships of DENV-2 from Central America, and report the first fully sequenced DENV-2 strain from Guatemala. Our analysis of the envelope (E) protein and of the open reading frame of strains from Central American countries, between 1999 and 2009, revealed that at least two lineages of the American/Asian genotype of DENV-2 have recently circulated in that region. In occasions the co-circulation of these lineages may have occurred and that has been suggested to play a role in the observed increased severity of clinical cases. Our time-scale analysis indicated that the most recent common ancestor for Central American DENV-2 of the American/Asian genotype existed about 19 years ago. Finally, we report positive selection in DENV-2 from Central America in codons of the genes encoding for C, E, NS2A, NS3, and NS5 proteins. Some of these identified codons are novel findings, described for the first time for any of the DENV-2 genotypes.     

Origin and evolution of dengue virus type 3 in Brazil

The incidence of dengue fever and dengue hemorrhagic fever in Brazil experienced a significant increase since the emergence of dengue virus type-3 (DENV-3) at the early 2000s. Despite the major public health concerns, there have been very few studies of the molecular epidemiology and time-scale of this DENV lineage in Brazil. In this study, we investigated the origin and dispersion dynamics of DENV-3 genotype III in Brazil by examining a large number (n = 107) of E gene sequences sampled between 2001 and 2009 from diverse Brazilian regions. These Brazilian sequences were combined with 457 DENV-3 genotype III E gene sequences from 29 countries around the world. Our phylogenetic analysis reveals that there have been at least four introductions of the DENV-3 genotype III in Brazil, as signified by the presence of four phylogenetically distinct lineages. Three lineages (BR-I, BR-II, and BR-III) were probably imported from the Lesser Antilles (Caribbean), while the fourth one (BR-IV) was probably introduced from Colombia or Venezuela. While lineages BR-I and BR-II succeeded in getting established and disseminated in Brazil and other countries from the Southern Cone, lineages BR-III and BR-IV were only detected in one single individual each from the North region. The phylogeographic analysis indicates that DENV-3 lineages BR-I and BR-II were most likely introduced into Brazil through the Southeast and North regions around 1999 (95% HPD: 1998–2000) and 2001 (95% HPD: 2000–2002), respectively. These findings show that importation of DENV-3 lineages from the Caribbean islands into Brazil seems to be relatively frequent. Our study further suggests that the North and Southeast Brazilian regions were the most important hubs of introduction and spread of DENV-3 lineages and deserve an intense epidemiological surveillance.     

Co-circulation of two genotypes of Dengue virus serotype 3 in Guangzhou, China, 2009

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.     

Evolutionary Analysis of Dengue Serotype 2 Viruses Using Phylogenetic and Bayesian Methods from New Delhi,India

Dengue fever is the most important arboviral disease in the tropical and sub-tropical countries of the world. Delhi, the metropolitan capital state of India, has reported many dengue outbreaks, with the last outbreak occurring in 2013. We have recently reported predominance of dengue virus serotype 2 during 2011–2014 in Delhi. In the present study, we report molecular characterization and evolutionary analysis of dengue serotype 2 viruses which were detected in 2011–2014 in Delhi. Envelope genes of 42 DENV-2 strains were sequenced in the study. All DENV-2 strains grouped within the Cosmopolitan genotype and further clustered into three lineages; Lineage I, II and III. Lineage III replaced lineage I during dengue fever outbreak of 2013. Further, a novel mutation Thr404Ile was detected in the stem region of the envelope protein of a single DENV-2 strain in 2014. Nucleotide substitution rate and time to the most recent common ancestor were determined by molecular clock analysis using Bayesian methods. A change in effective population size of Indian DENV-2 viruses was investigated through Bayesian skyline plot. The study will be a vital road map for investigation of epidemiology and evolutionary pattern of dengue viruses in India.     

Isolation and characterization of dengue virus serotype 2 from the large dengue outbreak in Guangdong,China in 2014

Dengue has been well recognized as a global public health threat,but only sporadic epidemics and imported cases were reported in recent decades in China.Since July 2014,an unexpected large dengue outbreak has occurred in Guangdong province,China,resulting in more than 40000 patients including six deaths.To clarify and characterize the causative agent of this outbreak,the acute phase serum from a patient diagnosed with severe dengue was subjected to virus isolation and high-throughput sequencing(HTS).Traditional real-time RT-PCR and HTS with Ion Torrent PGM detected the presence of dengue virus serotype 2(DENV-2).A clinical DENV-2 isolate GZ05/2014 was obtained by culturing the patient serum in mosquito C6/36 cells.The complete genome of GZ05/2014 was determined and deposited in Gen Bank under the access number KP012546.Phylogenetic analysis based on the complete envelope gene showed that the newly DENV-2 isolate belonged to Cosmopolitan genotype and clustered closely with other Guangdong strains isolated in the past decade.No amino acid mutations that are obviously known to increase virulence or replication were identified throughout the genome of GZ05/2014.The high homology of Guangdong DENV-2 strains indicated the possibility of establishment of local DENV-2 circulation in Guangdong,China.These results help clarify the origin of this epidemic and predict the future status of dengue in China.     

Pylogenetic relationship of dengue virus type 3 isolated in Brazil and Paraguay and global evolutionary divergence dynamics

ABSTRACT: BACKGROUND: Dengue is the most important mosquito-borne viral disease worldwide. Dengue virus comprises four antigenically related viruses named dengue virus type 1 to 4 (DENV1-4). DENV-3 was re-introduced into the Americas in 1994 causing outbreaks in Nicaragua and Panama. DENV-3 was introduced in Brazil in 2000 and then spread to most of the Brazilian States, reaching the neighboring country, Paraguay in 2002. In this study, we have analyzed the phylogenetic relationship of DENV-3 isolated in Brazil and Paraguay with viruses isolated worldwide. We have also analyzed the evolutionary divergence dynamics of DENV-3 viruses. RESULTS: The entire open reading frame (ORF) of thirteen DENV-3 isolated in Brazil (n= 9) and Paraguay (n= 4) were sequenced for phylogenetic analysis. DENV-3 grouped into three main genotypes (I, II and III). Several internal clades were found within each genotype that we called lineage and sub-lineage. Viruses included in this study belong to genotype III and grouped together with viruses isolated in the Americas within the lineage III. The Brazilian viruses were further segregated into two different sub-lineage, A and B, and the Paraguayan into the sub-lineage B. All three genotypes showed internal grouping. The nucleotide divergence was in average 6.7% for genotypes, 2.7% for lineages and 1.5% for sub-lineages. Phylogenetic trees constructed with any of the protein gene sequences showed the same segregation of the DENV-3 in three genotypes. CONCLUSION: Our results showed that two groups of DENV-3 genotypes III circulated in Brazil during 2002-2009, suggesting different events of introduction of the virus through different regions of the country. In Paraguay, only one group DENV-3 genotype III is circulating that is very closely related to the Brazilian viruses of sub-lineage B. Different degree of grouping can be observed for DENV-3 and each group showed a characteristic evolutionary divergence. Finally, we have observed that any protein gene sequence can be used to identify the virus genotype.     

Immunological and viral determinants of dengue severity in hospitalized adults in Ha Noi, Viet Nam

Background

The relationships between the infecting dengue serotype, primary and secondary infection, viremia and dengue severity remain unclear. This cross-sectional study examined these interactions in adult patients hospitalized with dengue in Ha Noi.

Methods and Findings

158 patients were enrolled between September 16 and November 11, 2008. Quantitative RT-PCR, serology and NS1 detection were used to confirm dengue infection, determine the serotype and plasma viral RNA concentration, and categorize infections as primary or secondary. 130 (82%) were laboratory confirmed. Serology was consistent with primary and secondary infection in 34% and 61%, respectively. The infecting serotype was DENV-1 in 42 (32%), DENV-2 in 39 (30%) and unknown in 49 (38%). Secondary infection was more common in DENV-2 infections (79%) compared to DENV-1 (36%, p<0.001). The proportion that developed dengue haemorrhagic fever (DHF) was 32% for secondary infection compared to 18% for primary infection (p = 0.14), and 26% for DENV-1 compared to 28% for DENV-2. The time until NS1 and plasma viral RNA were undetectable was shorter for DENV-2 compared to DENV-1 (p≤0.001) and plasma viral RNA concentration on day 5 was higher for DENV-1 (p = 0.03). Plasma viral RNA concentration was higher in secondary infection on day 5 of illness (p = 0.046). We didn''t find an association between plasma viral RNA concentration and clinical severity.

Conclusion

Dengue is emerging as a major public health problem in Ha Noi. DENV-1 and DENV-2 were the prevalent serotypes with similar numbers and clinical presentation. Secondary infection may be more common amongst DENV-2 than DENV-1 infections because DENV-2 infections resulted in lower plasma viral RNA concentrations and viral RNA concentrations were higher in secondary infection. The drivers of dengue emergence in northern Viet Nam need to be elucidated and public health measures instituted.     

Phylogeography of Recently Emerged DENV-2 in Southern Viet Nam

Revealing the dispersal of dengue viruses (DENV) in time and space is central to understanding their epidemiology. However, the processes that shape DENV transmission patterns at the scale of local populations are not well understood, particularly the impact of such factors as human population movement and urbanization. Herein, we investigated trends in the spatial dynamics of DENV-2 transmission in the highly endemic setting of southern Viet Nam. Through a phylogeographic analysis of 168 full-length DENV-2 genome sequences obtained from hospitalized dengue cases from 10 provinces in southern Viet Nam, we reveal substantial genetic diversity in both urban and rural areas, with multiple lineages identified in individual provinces within a single season, and indicative of frequent viral migration among communities. Focusing on the recently introduced Asian I genotype, we observed particularly high rates of viral exchange between adjacent geographic areas, and between Ho Chi Minh City, the primary urban center of this region, and populations across southern Viet Nam. Within Ho Chi Minh City, patterns of DENV movement appear consistent with a gravity model of virus dispersal, with viruses traveling across a gradient of population density. Overall, our analysis suggests that Ho Chi Minh City may act as a source population for the dispersal of DENV across southern Viet Nam, and provides further evidence that urban areas of Southeast Asia play a primary role in DENV transmission. However, these data also indicate that more rural areas are also capable of maintaining virus populations and hence fueling DENV evolution over multiple seasons.     

Re-Emergence of Dengue Virus Type 3 in Canton,China, 2009–2010, Associated with Multiple Introductions through Different Geographical Routes

Background

Endemic dengue virus type 3 (DENV-3) infections have not been reported in Canton, China, since 1980. In March 2009, DENV-3 was isolated for the second time, occurring about 30 years after the previous circulation. In August, 3 other cases emerged. One much larger outbreak occurred again in 2010. To address the origin and particularly to determine whether the outbreaks were caused by the same viral genotype, we investigated the epidemiological and molecular characteristics of the introduction, spread and genetic microevolution of DENV-3 involved.

Methodology/Principal Findings

Three imported cases (index-1,2,3) separately traveled back from Vietnam, India and Tanzania, resulted in 1, 3 and 60 secondary autochthonous cases, respectively. In autochthonous cases, 64.6% positive in IgM anti-DENV and 18.6% in IgG from a total of 48 submitted serum samples, accompanied by 7 DENV-3 isolates. With 99.8%, 99.7%, and 100% envelope gene nucleotidic identity, 09/GZ/1081 from index-1 and endemic strain (09/GZ/1483) belonged to genotype V; 09/GZ/10616 from index-2 and endemic strains (09/GZ/11144 and 09/GZ/11194) belonged to genotype III Clade-A; and 10/GZ/4898 from index-3 and all four 2010 endemic DENV-3 strains belonged to genotype III Clade-B, respectively.

Conclusions/Significance

Both epidemiological and phylogenetic analyses showed that the 2010 outbreak of dengue was not a reemergence of the 2009 strain. Introductions of different genotypes following more than one route were important contributory factors for the 2009–2010 dengue epidemics/outbreaks in Canton. These findings underscore the importance of early detection and case management of imported case in preventing large-scale dengue epidemics among indigenous peoples of Canton.     

Emergence of dengue virus serotype 2 in Mauritania and molecular characterization of its circulation in West Africa

The number of sporadic and epidemic dengue fever cases have reportedly been increasing in recent years in some West African countries, such as Senegal and Mali. The first epidemic of laboratory-confirmed dengue occurred in Nouakchott, the capital city of Mauritania situated in the Saharan desert, in 2014. On-site diagnosis of dengue fever was established using a rapid diagnostic test for dengue. In parallel, the presence of Aedes aegypti mosquitoes in the city was confirmed. The initial diagnosis was confirmed by RT-PCR, which showed that all samples from the 2014 dengue epidemic in Nouakchott were dengue virus serotype 2 (DENV-2). The whole genome or envelope protein gene of these strains, together with other DENV-2 strains obtained from travelers returning from West African countries to France between 2016 and 2019 (including two Mauritanian strains in 2017 and 2018), were sequenced. Phylogenetic analysis suggested a recent emergence of an epidemic strain from the cosmopolitan genotype belonging to West African cosmopolitan lineage II, which is genetically distinct from African sylvatic genotype. The origin of this DENV-2 lineage is still unknown, but our data seem to suggest a recent and rapid dispersion of the epidemic strain throughout the region. More complete genome sequences of West African DENV-2 are required for a better understanding of the dynamics of its circulation. Arboviral surveillance and outbreak forecasting are urgently needed in West Africa.     

Emergence and Diversification of Dengue 2 Cosmopolitan Genotype in Pakistan, 2011

Major dengue epidemics have been observed in the Indian subcontinent since the 1980s and have occurred with increased hospitalizations and mortality. In 2011, the first major epidemic of dengue occurred in Lahore, the second largest city in Pakistan, and resulted in 21,685 confirmed cases and 350 deaths. To investigate the possible viral causes for the increased epidemic activity, we determined the predominant serotype and characterized the viruses genetically. Of 50 patients carefully selected as probable dengue fever or dengue hemorrhagic fever, 34 were positive by virologic testing (i.e. PCR and/or virus isolation). DENV-2 was detected in 32 patients and DENV-1 in two. A total of 24 partial and three full DENV genomes were sequenced. Phylogenetic analyses of the capsid (C), pre-membrane (prM), and envelope genes comprising 2500 nucleotides in length indicated that all DENV-2 isolates in Pakistan since 2007 form a monophyletic lineage that is endemic in the country. These viruses were all of the cosmopolitan genotype (IV) and most closely related to viruses isolated in India and Sri Lanka in the past two decades. Phylogenetic analyses of data currently available in GenBank suggest that the Cosmopolitan genotype has diverged into two geographically distinct sub-lineages: sub-lineage IV-a has only been observed in Southeast Asia, China and Oceania, while IV-b is prevalent in the Indian subcontinent. These results highlight the increased diversity of dengue viruses as they spread geographically within the region.     

Dengue Virus 2 American-Asian Genotype Identified during the 2006/2007 Outbreak in Piauí, Brazil Reveals a Caribbean Route of Introduction and Dissemination of Dengue Virus in Brazil

Dengue virus (DENV) is the most widespread arthropod-borne virus, and the number and severity of outbreaks has increased worldwide in recent decades. Dengue is caused by DENV-1, DENV- 2, DENV-3 and DENV-4 which are genetically distant. The species has been subdivided into genotypes based on phylogenetic studies. DENV-2, which was isolated from dengue fever patients during an outbreak in Piaui, Brazil in 2006/2007 was analyzed by sequencing the envelope (E) gene. The results indicated a high similarity among the isolated viruses, as well as to other DENV-2 from Brazil, Central America and South America. A phylogenetic and phylogeographic analysis based on DENV-2E gene sequences revealed that these viruses are grouped together with viruses of the American-Asian genotype in two distinct lineages. Our results demonstrate the co-circulation of two American-Asian genotype lineages in northeast Brazil. Moreover, we reveal that DENV-2 lineage 2 was detected in Piauí before it disseminated to other Brazilian states and South American countries, indicating the existence of a new dissemination route that has not been previously described.     

First Insights into the Phylogenetic Diversity of Mycobacterium tuberculosis in Nepal

Background

Tuberculosis (TB) is a major public health problem in Nepal. Strain variation in Mycobacterium tuberculosis may influence the outcome of TB infection and disease. To date, the phylogenetic diversity of M. tuberculosis in Nepal is unknown.

Methods and Findings

We analyzed 261 M. tuberculosis isolates recovered from pulmonary TB patients recruited between August 2009 and August 2010 in Nepal. M. tuberculosis lineages were determined by single nucleotide polymorphisms (SNP) typing and spoligotyping. Drug resistance was determined by sequencing the hot spot regions of the relevant target genes. Overall, 164 (62.8%) TB patients were new, and 97 (37.2%) were previously treated. Any drug resistance was detected in 50 (19.2%) isolates, and 16 (6.1%) were multidrug-resistant. The most frequent M. tuberculosis lineage was Lineage 3 (CAS/Delhi) with 106 isolates (40.6%), followed by Lineage 2 (East-Asian lineage, includes Beijing genotype) with 84 isolates (32.2%), Lineage 4 (Euro-American lineage) with 41 (15.7%) isolates, and Lineage 1 (Indo-Oceanic lineage) with 30 isolates (11.5%). Based on spoligotyping, we found 45 different spoligotyping patterns that were previously described. The Beijing (83 isolates, 31.8%) and CAS spoligotype (52, 19.9%) were the dominant spoligotypes. A total of 36 (13.8%) isolates could not be assigned to any known spoligotyping pattern. Lineage 2 was associated with female sex (adjusted odds ratio [aOR] 2.58, 95% confidence interval [95% CI] 1.42–4.67, p = 0.002), and any drug resistance (aOR 2.79; 95% CI 1.43–5.45; p = 0.002). We found no evidence for an association of Lineage 2 with age or BCG vaccination status.

Conclusions

We found a large genetic diversity of M. tuberculosis in Nepal with representation of all four major lineages. Lineages 3 and 2 were dominating. Lineage 2 was associated with clinical characteristics. This study fills an important gap on the map of the M. tuberculosis genetic diversity in the Asian region.     

Invasion and maintenance of dengue virus type 2 and type 4 in the Americas

Dengue virus type 4 (DENV-4) was first reported in the Americas in 1981, where it caused epidemics of dengue fever throughout the region. In the same year, the region's first epidemic of dengue hemorrhagic fever was reported, caused by an Asian strain of dengue virus type 2 (DENV-2) that was distinct from the American subtype circulating previously. Despite the importance of these epidemics, little is known about the rates or determinants of viral spread among island and mainland populations or their directions of movement. We employed a Bayesian coalescent approach to investigate the transmission histories of DENV-2 and DENV-4 since their introduction in 1981 and a parsimony method to assess patterns of strain migration. For both viruses there was an initial invasion phase characterized by an exponential increase in the number of DENV lineages, after which levels of genetic diversity remained constant despite reported fluctuations in DENV-2 and DENV-4 activity. Strikingly, viral lineage numbers increased far more rapidly for DENV-4 than DENV-2, indicative of a more rapid rate of exponential population growth in DENV-4 or a higher rate of geographic dispersal, allowing this virus to move more effectively among localities. We propose that these contrasting dynamics may reflect underlying differences in patterns of host immunity. Despite continued gene flow along particular transmission routes, the overall extent of viral traffic was less than expected under panmixis. Hence, DENV in the Americas has a clear geographic structure that maintains viral diversity between outbreaks.     

Phylogeography and population dynamics of dengue viruses in the Americas

Changes in Dengue virus (DENV) disease patterns in the Americas over recent decades have been attributed, at least in part, to repeated introduction of DENV strains from other regions, resulting in a shift from hypoendemicity to hyperendemicity. Using newly sequenced DENV-1 and DENV-3 envelope (E) gene isolates from 11 Caribbean countries, along with sequences available on GenBank, we sought to document the population genetic and spatiotemporal transmission histories of the four main invading DENV genotypes within the Americas and investigate factors that influence the rate and intensity of DENV transmission. For all genotypes, there was an initial invasion phase characterized by rapid increases in genetic diversity, which coincided with the first confirmed cases of each genotype in the region. Rapid geographic dispersal occurred upon each genotype's introduction, after which individual lineages were locally maintained, and gene flow was primarily observed among neighboring and nearby countries. There were, however, centers of viral diversity (Barbados, Puerto Rico, Colombia, Suriname, Venezuela, and Brazil) that were repeatedly involved in gene flow with more distant locations. For DENV-1 and DENV-2, we found that a "distance-informed" model, which posits that the intensity of virus movement between locations is inversely proportional to the distance between them, provided a better fit than a model assuming equal rates of movement between all pairs of countries. However, for DENV-3 and DENV-4, the more stochastic "equal rates" model was preferred.