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. 2018 Nov 12;13(11):e0207220.
doi: 10.1371/journal.pone.0207220. eCollection 2018.

Emergence of genotype Cosmopolitan of dengue virus type 2 and genotype III of dengue virus type 3 in Thailand

Juthamas Phadungsombat  1 , Marco Yung-Cheng Lin  2 , Narinee Srimark  1 , Atsushi Yamanaka  1 , Emi E Nakayama  1   3 , Visal Moolasart  4 , Patama Suttha  4 , Tatsuo Shioda  1   3 , Sumonmal Uttayamakul  4
Affiliations

Emergence of genotype Cosmopolitan of dengue virus type 2 and genotype III of dengue virus type 3 in Thailand

Juthamas Phadungsombat et al. PLoS One. .

Abstract

Dengue is a mosquito-borne disease that has spread to over 100 countries. Dengue fever is caused by dengue virus (DENV), which belongs to the Flavivirus genus of the family Flaviviridae. DENV comprises 4 serotypes (DENV-1 to DENV-4), and each serotype is divided into distinct genotypes. Thailand is an endemic area where all 4 serotypes of DENV co-circulate. To understand the current genotype distribution of DENVs in Thailand, we enrolled 100 cases of fever with dengue-like symptoms at the Bamrasnaradura Infectious Diseases Institute during 2016-2017. Among them, 37 cases were shown to be dengue-positive by real-time PCR. We were able to isolate DENVs from 21 cases, including 1 DENV-1, 8 DENV-2, 4 DENV-3, and 8 DENV-4. To investigate the divergence of the viruses, RNA was extracted from isolated DENVs and viral near-whole genome sequences were determined. Phylogenetic analysis of the obtained viral sequences revealed that DENV-2 genotype Cosmopolitan was co-circulating with DENV-2 genotype Asian-I, the previously predominating genotype in Thailand. Furthermore, DENV-3 genotype III was found instead of DENV-3 genotype II. The DENV-2 Cosmopolitan and DENV-3 genotype III found in Thailand were closely related to the respective strains found in nearby countries. These results indicated that DENVs in Thailand have increased in genotypic diversity, and suggested that the DENV genotypic shift observed in other Asian countries also might be taking place in Thailand.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Dengue virus genotyping.
The maximum likelihood phylogenetic tree was constructed in the IQ-TREE program using the general time reversible model with gamma distribution and invariant sites with 500 bootstrap replications. Data included envelope-encoding sequences obtained in the present study (labeled in red) along with sequences obtained from GenBank. The viral genotypes are indicated to the right. Virus names are shown as accession number, country, and reported year of each sequence. Numbers on branches are bootstrap support values exceeding 75%.
Fig 2
Fig 2. Phylogeny of Thailand DENV-2 strains.
The maximum-likelihood phylogenetic tree was constructed in IQ-TREE version 1.6.7 using the TN+F+G4 with 500 bootstrap replications. Data included envelope-encoding sequences obtained in the present study (labeled in red) along with sequences obtained from Genbank (Asian-I, black; Cosmopolitan, blue). The viral genotypes are indicated to the right. Virus names are shown as accession number, country, and reported year of each sequence. Numbers on branches are bootstrap support values exceeding 75%.
Fig 3
Fig 3. Molecular clock analysis of DENV-2 genotype Asian-I envelope-encoding sequences.
(a) Correlation of collection year and divergence from the maximum likelihood tree. The R2 (coefficient of determination) of 0.79 was estimated using TempEst (shown at the top left). Red dots indicate sequences obtained in the present study. (b) Bayesian maximum clade credibility (MCC) phylogenetic tree estimated using BEAST v1.8.4. The terminal branch color indicates different geographical locations according to the legend at the bottom left corner of the figure. The mean time of the most recent common ancestor (tMRCA) and 95% highest probability density (HPD) (in calendar year and tenths of year) are indicated with a black arrow, and posterior probability values are indicated adjacent to the node of interest. The name of each taxon is presented formatted as accession number, country, and year of collection. Sequences obtained in the present study are labeled in red. (c) Demographic history of DENV-2 genotype Asian-I was inferred by a Gaussian Markov random field (GMRF) Skyride plot using Tracer 1.7.
Fig 4
Fig 4. Molecular clock analysis of DENV-2 genotype Cosmopolitan envelope-encoding sequences.
(a) Correlation of collection year and divergence from maximum likelihood tree. The R2, (coefficient of determination) of 0.82 was estimated using TempEst (shown at the top left). Red dots indicate sequences obtained in the present study. (b) Bayesian maximum clade credibility (MCC) phylogenetic tree estimated using BEAST v1.8.4. The terminal branch color indicates different geographical locations according to legend at the bottom left corner of the figure. The mean time of the most recent common ancestor (tMRCA) and 95% highest probability density (HPD) (in calendar year and tenths of year) are indicated with black arrows, and posterior probability values are indicated adjacent to the node of interest. The name of each taxon is presented formatted as accession number, country, and year of collection. Sequences obtained in the present study are labeled in red, while Thailand sequences obtained from GenBank are labeled in blue. Lineages A, B, and C are shown to the right. The KX621247 virus is indicated by a red asterisk. (c) Demographic history of DENV-2 genotype Cosmopolitan was inferred by a Gaussian Markov random field (GMRF) Skyride plot using Tracer 1.7.
Fig 5
Fig 5. Phylogeny of Thailand DENV-3 strains.
The maximum likelihood phylogenetic tree was constructed in IQ-TREE version 1.6.7 using the TN+F+G4 with 500 bootstrap replications. Data included envelope-encoding sequences obtained in the present study (labeled in red) along with sequences obtained from Genbank (genotype II, black; genotype III, blue). The viral genotypes are indicated to the right. Virus names are shown as accession number, country, and reported year of each sequence. Numbers on branches are bootstrap support values exceeding 75%.
Fig 6
Fig 6
Molecular clock analysis of DENV-3 genotype III envelope-encoding sequences (a) Correlation of collection year and divergence from maximum likelihood tree. The R2 (coefficient of determination) of 0.78 was estimated using TempEst (shown at top left). Red dots indicate sequences obtained in the present study. (b) Bayesian maximum clade credibility (MCC) phylogenetic tree estimated using BEAST v1.8.4. The terminal branch color indicates different geographical locations according to the legend at the bottom left corner of the figure. The mean time of the most recent common ancestor (tMRCA) and 95% highest probability density (HPD) (in calendar year and tenths of year) are indicated with black arrows, and posterior probability values are indicated adjacent to the node of interest. The name of each taxon is presented formatted as accession number, country, and year of collection. Sequences obtained in the present study are labeled in red, while Thailand sequences from GenBank are labeled in blue. Lineages A, B, and C are shown to the right. The JQ922556 virus is indicated by a red asterisk. (c) Demographic history of DENV-3 genotype III was inferred by a Gaussian Markov random field (GMRF) Skyride plot using Tracer 1.7.
Fig 7
Fig 7. Amino acid variations among ORF proteins encoded by DENV-2 genotype Cosmopolitan.
The amino acid substitutions in the open reading frames of the 3 lineages of DENV-2 genotype Cosmopolitan (in the right panel) are shown corresponding to the lineages. The occurrences of indicated amino acid variations are shown with arrows. Sequences obtained in the present study are labeled in red.
Fig 8
Fig 8. Amino acid variations among ORF proteins encoded by DENV-3 genotype III.
The amino acid substitutions in the open reading frame among 3 lineages of DENV-3 genotype III (in the right panel) are shown corresponding to the lineages. The occurrences of the indicated amino acid variations are shown with arrows. Sequences obtained in this study are labeled in red.
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Grants and funding

This work was supported by funder Japan Agency for Medical Research and Development (AMED), https://www.amed.go.jp/; Program: Japan Initiative for Global Research Network on Infectious Diseases (J-GRID) JP18fm0108003 to Tatsuo Shioda.