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.     

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.     

Phylogenetic and Molecular Clock Analysis of Dengue Serotype 1 and 3 from New Delhi,India

Dengue fever is the most prevalent arboviral disease in the tropical and sub-tropical regions of the world. The present report describes molecular detection and serotyping of dengue viruses in acute phase blood samples collected from New Delhi, India. Phylogenetic and molecular clock analysis of dengue virus serotype 1 and 3 strains were also investigated. Dengue virus infection was detected in 68.87% out of 604 samples tested by RT-PCR between 2011 & 2014. Dengue serotype 1 was detected in 25.48% samples, dengue serotype 2 in 79.56% samples and dengue serotype 3 in 11.29% samples. Dengue serotype 4 was not detected. Co-infection by more than one dengue serotype was detected in 18.26% samples. Envelope gene of 29 DENV-1 and 14 DENV-3 strains were sequenced in the study. All the DENV-1 strains grouped with the American African genotype. All DENV-3 strains were found to belong to Genotype III. Nucleotide substitution rates of dengue 1 and 3 viruses were determined in the study. Time to the most recent common ancestor (TMRCA) of dengue 1 viruses was determined to be 132 years. TMRCA of DENV-3 viruses was estimated to be 149 years. Bayesian skyline plots were constructed for Indian DENV-1 and 3 strains which showed a decrease in population size since 2005 in case of DENV- 1 strains while no change was observed in recent years in case of DENV-3 strains. The study also revealed a change in the dominating serotype in Delhi, India in recent years. The study will be helpful in formulating control strategies for the outbreaks. In addition, it will also assist in tracking the movement and evolution of this emerging virus.     

Air Travel Is Associated with Intracontinental Spread of Dengue Virus Serotypes 1–3 in Brazil

Dengue virus and its four serotypes (DENV-1 to DENV-4) infect 390 million people and are implicated in at least 25,000 deaths annually, with the largest disease burden in tropical and subtropical regions. We investigated the spatial dynamics of DENV-1, DENV-2 and DENV-3 in Brazil by applying a statistical framework to complete genome sequences. For all three serotypes, we estimated that the introduction of new lineages occurred within 7 to 10-year intervals. New lineages were most likely to be imported from the Caribbean region to the North and Northeast regions of Brazil, and then to disperse at a rate of approximately 0.5 km/day. Joint statistical analysis of evolutionary, epidemiological and ecological data indicates that aerial transportation of humans and/or vector mosquitoes, rather than Aedes aegypti infestation rates or geographical distances, determine dengue virus spread in Brazil.     

Studies on membrane topology, N-glycosylation and functionality of SARS-CoV membrane protein

Background

Dengue is the most important arbovirus disease in tropical and subtropical countries. The viral envelope (E) protein is responsible for cell receptor binding and is the main target of neutralizing antibodies. The aim of this study was to analyze the diversity of the E protein gene of DENV-3. E protein gene sequences of 20 new viruses isolated in Ribeirao Preto, Brazil, and 427 sequences retrieved from GenBank were aligned for diversity and phylogenetic analysis.

Results

Comparison of the E protein gene sequences revealed the presence of 47 variable sites distributed in the protein; most of those amino acids changes are located on the viral surface. The phylogenetic analysis showed the distribution of DENV-3 in four genotypes. Genotypes I, II and III revealed internal groups that we have called lineages and sub-lineages. All amino acids that characterize a group (genotype, lineage, or sub-lineage) are located in the 47 variable sites of the E protein.

Conclusion

Our results provide information about the most frequent amino acid changes and diversity of the E protein of DENV-3.     

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.     

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.     

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.     

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.     

A novel virus that infecting hypovirulent strain XG36-1 of plant fungal pathogen Sclerotinia sclerotiorum

Background

Dengue virus type 1 (DENV-1) have been mostly circulating silently with dominant serotypes DENV-2 and DENV-3 in India. However recent times have marked an increase in DENV-1 circulation in yearly outbreaks. Many studies have not been carried out on this virus type, leaving a lacunae pertaining to the circulating genotypes, since its earliest report in India. In the present study, we sequenced CprM gene junction of 13 DENV-1 isolated from Delhi and Gwalior (North India) between 2001–2007 and one 1956 Vellore isolate as reference. For comparison, we retrieved 11 other Indian and 70 global reference sequences from NCBI database, making sure that Indian and global isolates from all decades are available for comparative analysis.

Results

The region was found to be AT rich with no insertion or deletion. Majority of the nucleotide substitutions were silent, except 3 non-conservative amino acid changes (I → T, A → T and L → S at amino acid positions 59,114 and 155 respectively) in the Indian DENV-1 sequences, sequenced in this study. Except two 1997–98 Delhi isolates, which group in genotype I; all other Indian isolates group in genotype III. All Indian genotype III DENV-1 exhibited diversity among them, giving rise to at least 4 distinct lineages (India 1–4) showing proximity to isolates from diverse geographic locations.

Conclusion

The extensive phylogenetic analysis revealed consistent existence of multiple lineages of DENV-1 genotype III during the last 5 decades in India.     

The Complete Genome Phylogeny of Geographically Distinct Dengue Virus Serotype 2 Isolates (1944-2013) Supports Further Groupings within the Cosmopolitan Genotype

Dengue virus serotype 2 (DENV-2) isolates have been implicated in deadly outbreaks of dengue fever (DF) and dengue hemorrhagic fever (DHF) in several regions of the world. Phylogenetic analysis of DENV-2 isolates collected from particular countries has been performed using partial or individual genes but only a few studies have examined complete whole-genome sequences collected worldwide. Herein, 50 complete genome sequences of DENV-2 isolates, reported over the past 70 years from 19 different countries, were downloaded from GenBank. Phylogenetic analysis was conducted and evolutionary distances of the 50 DENV-2 isolates were determined using maximum likelihood (ML) trees or Bayesian phylogenetic analysis created from complete genome nucleotide (nt) and amino acid (aa) sequences or individual gene sequences. The results showed that all DENV-2 isolates fell into seven main groups containing five previously defined genotypes. A Cosmopolitan genotype showed further division into three groups (C-I, C-II, and C-III) with the C-I group containing two subgroups (C-IA and C-IB). Comparison of the aa sequences showed specific mutations among the various groups of DENV-2 isolates. A maximum number of aa mutations was observed in the NS5 gene, followed by the NS2A, NS3 and NS1 genes, while the smallest number of aa substitutions was recorded in the capsid gene, followed by the PrM/M, NS4A, and NS4B genes. Maximum evolutionary distances were found in the NS2A gene, followed by the NS4A and NS4B genes. Based on these results, we propose that genotyping of DENV-2 isolates in future studies should be performed on entire genome sequences in order to gain a complete understanding of the evolution of various isolates reported from different geographical locations around the world.     

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.     

Evaluation of the traditional and revised WHO classifications of Dengue disease severity

Dengue is a major public health problem worldwide and continues to increase in incidence. Dengue virus (DENV) infection leads to a range of outcomes, including subclinical infection, undifferentiated febrile illness, Dengue Fever (DF), life-threatening syndromes with fluid loss and hypotensive shock, or other severe manifestations such as bleeding and organ failure. The long-standing World Health Organization (WHO) dengue classification and management scheme was recently revised, replacing DF, Dengue Hemorrhagic Fever (DHF), and Dengue Shock Syndrome (DSS) with Dengue without Warning Signs, Dengue with Warning Signs (abdominal pain, persistent vomiting, fluid accumulation, mucosal bleeding, lethargy, liver enlargement, increasing hematocrit with decreasing platelets) and Severe Dengue (SD; dengue with severe plasma leakage, severe bleeding, or organ failure). We evaluated the traditional and revised classification schemes against clinical intervention levels to determine how each captures disease severity using data from five years (2005–2010) of a hospital-based study of pediatric dengue in Managua, Nicaragua. Laboratory-confirmed dengue cases (n = 544) were categorized using both classification schemes and by level of care (I–III). Category I was out-patient care, Category II was in-patient care that did not meet criteria for Category III, which included ICU admission, ventilation, administration of inotropic drugs, or organ failure. Sensitivity and specificity to capture Category III care for DHF/DSS were 39.0% and 75.5%, respectively; sensitivity and specificity for SD were 92.1% and 78.5%, respectively. In this data set, DENV-2 was found to be significantly associated with DHF/DSS; however, this association was not observed with the revised classification. Among dengue-confirmed cases, the revised WHO classification for severe dengue appears to have higher sensitivity and specificity to identify cases in need of heightened care, although it is no longer as specific for a particular pathogenic entity as was the traditional schema.     

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.     

Evolution of endemic and sylvatic lineages of dengue virus

Recent disease outbreaks have raised awareness of tropical pathogens, especially mosquito-borne viruses. Dengue virus (DENV) is a widely studied mammalian pathogen transmitted by various species of mosquito in the genus Aedes, especially Aedes aegypti and Aedes albopictus. The prevailing view of the research community is that endemic viral lineages that cause epidemics of DENV in humans have emerged over time from sylvatic viral lineages, which persist in wild, non-human primates. These notions have been examined by researchers through phylogenetic analyses of the envelope gene (E) from the four serotypes of DENV (serotypes DENV-1 to DENV-4). In these previous reports, researchers used visual inspection of a phylogeny in order to assert that sylvatic lineages lead to endemic clades. In making this assertion, these researchers also reasserted the model of periodic sylvatic to endemic disease outbreaks. Since that study, there has been a significant increase in data both in terms of metadata (e.g., place and host of isolation) and genetic sequences of DENV. Here, we re-examine the model of sylvatic to endemic shifts in viral lineages through a phylogenetic tree search and character evolution study of metadata on the tree. We built a dataset of nucleotide sequences for 188 isolates of DENV that have metadata on sylvatic or endemic sampling along with three orthologous sequences from West Nile virus as the outgroup for the phylogenetic analysis. In contrast to previous research, we find that there are several shifts from endemic to sylvatic lineages as well as sylvatic to endemic lineages, indicating a much more dynamic model of evolution. We propose that a model that allows oscillation between sylvatic and endemic hosts better captures the dynamics of DENV transmission.     

Laboratory Surveillance of Dengue in Rio Grande do Sul,Brazil, from 2007 to 2013

Background

According to official records, dengue was introduced in Brazil in the 80''s; since then several epidemics have occurred. Meanwhile, in Rio Grande do Sul (RS, Southern Brazil) the first autochthonous case occurred only in 2007.

Methodology and Principal Findings

In this study we report laboratory surveillance of dengue cases and seasonality of positive cases, describe serotypes and characterize the epidemiological pattern of dengue in RS from 2007 to 2013. A total of 9,779 serum samples from patients with suspected dengue fever were collected and submitted to molecular and/or serological analyses for dengue virus identification and serotyping, based on viral isolation, NS1 antigen detection and qRT-PCR, or Dengue IgM capture ELISA and MAC-ELISA. The first autochthonous dengue case in RS was confirmed in 2007 (DENV-3). While in 2008 and 2009 only imported cases were registered, autochthonous infection waves have been occurring since 2010. The highest number of dengue infections occurred in 2010, with DENV-1 and DENV-2 outbreaks in Northwestern RS. In 2011, another DENV-1 and DENV-2 outbreak occurred in the Northwestern region; moreover, DENV-4 was detected in travelers. In 2012, DENV-1 and DENV-4 co-circulated. DENV-2 circulation was only detected again in 2013, in high frequency (56.7%), co-circulating with DENV-4 (35%). Most infections occur in adults during summer. Differences in prevalence between genders were observed in 2007 (60% females), 2008 (60.8% males) and 2009 (77.5% males).

Conclusions

According to results of dengue surveillance, there was an increase in the number of dengue cases in RS and of cities infested with Aedes aegypti, possibly as a consequence of introduction of new serotypes and the difficulty of health programs to control the vector.     

Spatio-Temporal Tracking and Phylodynamics of an Urban Dengue 3 Outbreak in S?o Paulo,Brazil

The dengue virus has a single-stranded positive-sense RNA genome of ∼10.700 nucleotides with a single open reading frame that encodes three structural (C, prM, and E) and seven nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins. It possesses four antigenically distinct serotypes (DENV 1–4). Many phylogenetic studies address particularities of the different serotypes using convenience samples that are not conducive to a spatio-temporal analysis in a single urban setting. We describe the pattern of spread of distinct lineages of DENV-3 circulating in São José do Rio Preto, Brazil, during 2006. Blood samples from patients presenting dengue-like symptoms were collected for DENV testing. We performed M-N-PCR using primers based on NS5 for virus detection and identification. The fragments were purified from PCR mixtures and sequenced. The positive dengue cases were geo-coded. To type the sequenced samples, 52 reference sequences were aligned. The dataset generated was used for iterative phylogenetic reconstruction with the maximum likelihood criterion. The best demographic model, the rate of growth, rate of evolutionary change, and Time to Most Recent Common Ancestor (TMRCA) were estimated. The basic reproductive rate during the epidemics was estimated. We obtained sequences from 82 patients among 174 blood samples. We were able to geo-code 46 sequences. The alignment generated a 399-nucleotide-long dataset with 134 taxa. The phylogenetic analysis indicated that all samples were of DENV-3 and related to strains circulating on the isle of Martinique in 2000–2001. Sixty DENV-3 from São José do Rio Preto formed a monophyletic group (lineage 1), closely related to the remaining 22 isolates (lineage 2). We assumed that these lineages appeared before 2006 in different occasions. By transforming the inferred exponential growth rates into the basic reproductive rate, we obtained values for lineage 1 of R₀ = 1.53 and values for lineage 2 of R₀ = 1.13. Under the exponential model, TMRCA of lineage 1 dated 1 year and lineage 2 dated 3.4 years before the last sampling. The possibility of inferring the spatio-temporal dynamics from genetic data has been generally little explored, and it may shed light on DENV circulation. The use of both geographic and temporally structured phylogenetic data provided a detailed view on the spread of at least two dengue viral strains in a populated urban area.     

Nature and Extent of Genetic Diversity of Dengue Viruses Determined by 454 Pyrosequencing

Dengue virus (DENV) populations are characteristically highly diverse. Regular lineage extinction and replacement is an important dynamic DENV feature, and most DENV lineage turnover events are associated with increased incidence of disease. The role of genetic diversity in DENV lineage extinctions is not understood. We investigated the nature and extent of genetic diversity in the envelope (E) gene of DENV serotype 1 representing different lineages histories. A region of the DENV genome spanning the E gene was amplified and sequenced by Roche/454 pyrosequencing. The pyrosequencing results identified distinct sub-populations (haplotypes) for each DENV-1 E gene. A phylogenetic tree was constructed with the consensus DENV-1 E gene nucleotide sequences, and the sequences of each constructed haplotype showed that the haplotypes segregated with the Sanger consensus sequence of the population from which they were drawn. Haplotypes determined through pyrosequencing identified a recombinant DENV genome that could not be identified through Sanger sequencing. Nucleotide level sequence diversities of DENV-1 populations determined from SNP analysis were very low, estimated from 0.009–0.01. There were also no stop codon, frameshift or non-frameshift mutations observed in the E genes of any lineage. No significant correlations between the accumulation of deleterious mutations or increasing genetic diversity and lineage extinction were observed (p>0.5). Although our hypothesis that accumulation of deleterious mutations over time led to the extinction and replacement of DENV lineages was ultimately not supported by the data, our data does highlight the significant technical issues that must be resolved in the way in which population diversity is measured for DENV and other viruses. The results provide an insight into the within-population genetic structure and diversity of DENV-1 populations.