Genotypes of Dengue Virus Circulate in Dengue Sentinel Surveillance in Indonesia

Background: Dengue hemorrhagic fever was caused by dengue virus (DENV). This virus has four serotypes, DENV-1 to DENV-4, in which each serotype consists of various genotypes. This study present the distribution of DENV genotype circulate in dengue sentinel surveillance sites. Methods: We performed molecular analyses using bioinformatics tools to identify genotype of DENV. Sequencing targeting envelope gene was carried out on DENV-positive samples. Full-length sequence of envelope gene was obtained using overlapping primers for DENV 1, 2, 3 and 4. Phylogenetic tree was generated by Neighbor Joining using MEGA 6.0 with 1000 bootstrap replications and Kimura 2-parameter model. Known genotype of DENV sequences from Indonesia and other countries obtained from GenBank were included in the analyses as references. Results: A total of 64 complete coding sequences of envelope gene from DENV-1, DENV-2 and DENV-3 were successfully sequenced from 204 confirmed DENV cases. There are fourteen sequences of DENV-1, 22 sequences of DENV-2 and 28 sequences of DENV-3. Unfortunately, there was no complete coding sequence of envelope gene obtained for DENV-4. DENV-1 and DENV-2 were grouped into genotype I and cosmopolitan genotype, respectively. The DENV-3 was grouped into genotype I. These viruses were belongs to various genotypes that have been circulating previously in Indonesia. Conclusion: This finding suggests that the distribution of DENV genotype in sentinel sites remained stable. (Health Science Journal of Indonesia 2016;7(2):69-74)

Dengue virus (DENV) is the causative agent of dengue infection. DENV belongs to family Flaviviridae and genus Flavivirus. DENV serotypes have four genetically and antigenically different, known as DENV-1, DENV-2, DENV-3 dan DENV-4. Each serotype is divided into several genotypes based on the sequence variability on the envelope gene (E gene). DENV-1 consists of ¿YH JHQRW\SHV JHQRW\SH , 9 1 DENV-2 comprises of American genotype, Cosmopolitan, Asian I, Asian II, American Asian genotype and Sylvatic. 2 DENV-3 consists of 4 genotypes (genotype I-IV) 3 and DENV-4 comprises of 4 genotypes (genotype I-III and sylvatic ). 4 *HQRW\SLQJ RI '(19 LV HVVHQWLDO DV WKH LGHQWL¿FDWLRQ of DENV genotype could provide information of genetic changes in DENV gene, and to detect the introduction of new genotype in certain area. In addition, DENV as RNA virus continues to evolve and WKH LGHQWL¿FDWLRQ RI '(19 JHQRW\SH FRXOG SURYLGH crucial information of the dynamic change of DENV. 5 It was reported that genotype I and IV of DENV-1 circulated in Indonesia. 6 So are cosmopolitan of DENV-2, genotype I of DENV-3 and genotype II of DENV-4. 7,8 Therefore limited data was available regarding the distribution of DENV genotype in dengue sentinel surveillance sites, which serum samples were sent to National Institute of Health Research and Development (NIHRD). The surveillance was conducted to obtain information on epidemiology and virology of DENV and other arboviruses. In this study we present the distribution of DENV genotype circulate in dengue sentinel surveillance sites.

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The overlapping nucleotide sequences were edited, assembled and aligned using BioEdit Sequence Alignment Editor Ver 7.0.5.2 . Multiple alignment of DENV envelope gene sequences (1485 nt for DENV-1 and DENV-2, 1479 nt for DENV-3) was performed by ClustalW using MEGA 6.0. 10 Then phylogenetic tree was generated by Neighbor Joining using the same software with 1000 bootstrap replicates and Kimura 2-parameter model. Existing genotype of DENV sequences from Indonesia and other countries obtained from GenBank were included in the analyses DV UHIHUHQFHV &ODVVL¿FDWLRQV VFKHPH RI JHQRW\SH utilized as proposed by previous publications. [1][2][3][4] The study got ethical approval from National Institute of Health Reseach and development ethical committee No. LB.02.01/5.2/KE. 208/2015.

RESULTS
During study period, 204 samples were DENV positive out of 585 specimens sent to NIHRD. All DENV serotypes, DENV-1 to DENV-4 were found from all of sentinels in which DENV-3 was the dominant serotypes.
A total of sixty four complete coding sequences of envelope gene from DENV-1, DENV-2 and, DENV-ZHUH VXFFHVVIXOO\ VHTXHQFHG IURP FRQ¿UPHG DENV cases. There was no complete coding sequence of envelope gene of DENV-4 obtained in this study. Figure 2 illustrated the phylogenetic tree of envelope gene of DENV-1 in which consisted of fourteen sequences of DENV-1, grouped in genotype I. The samples were originated from all of sentinel sites. The phylogenetic tree illustrated in Figure  2, shows that DENV-1 circulated in sentinel sites formed a cluster with those detected in Singapore 2007-2010. Nucleotide sequence identity between clades ranged from 97%-99%.
There were 22 samples that successfully sequenced for envelope gene of DENV-2. All sequences were grouped in cosmopolitan genotype. These sera samples came from all of sentinel site except Ambon. The DENV-2 phylogenetic tree illustrated in Figure 3, showed that the Indonesian Cosmopolitan genotype were closely related to isolates that circulated previously in Indonesia 7   In addition, 28 sequences of DENV-3 were grouped in genotype I. (Figure. 4)

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It was reported by Sjatha et.al (2012) that two genotypes of DENV-1 predominantly circulated in Indonesia, the genotype I and IV. The genotype IV of DENV-1 has been indigenous in Indonesia since 1948. It was likely to spread over the region and into the Indian Ocean countries and predicted diverged frequently around 1967. 6 In Surabaya, another city in which endemic of dengue, genotype of the DENV-1 was reported to shift from genotype IV to I in 2010. 12 The genotype I circulated in the sentinel sites, and it have been circulated in Medan and Bandung in 2008 (data not shown), but another report suggesting that this genotype has been circulated in Indonesia since 2005. 13 A recent study in Semarang has reported DENV-1 genotype II that was the old genotype circulated in Indonesia, 8 we did not ¿QG WKH JHQRW\SH ,, LQ WKLV VWXG\ The Cosmopolitan genotype of DENV-2 has a wide geographical distribution including strains from Southeast Asia, Australia, India, China and the Middle East. 14 Based on previous data, the Cosmopolitan genotype was separated into two geographically different sub-lineages. One sub-lineage observed in Southeast Asia, China and Oceania, other was observed in the India subcontinent. 15 The Cosmopolitan genotype circulated in sentinel sites found in this study, was VLPLODU ZLWK WKH ¿UVW VXE OLQHDJH  Genotype II Genotype IV DENV-3 is the most dominant serotypes found in this study and was grouped as genotype I. There were two sub-lineages of DENV-3 circulated in Indonesia that isolated before and after 1998, DENV-3 isolated after 1998 had gave greater impact on DHF epidemic. 16 We found that all DENV-3 sequences from sentinel sites had close related to DENV-3 isolated after 1998. Other author have observed the existence of some intragenotypic groups that reveal amino acid changes in the envelope gene of DENV-3. 17 6LQFH '(19 KDG VLJQL¿FDQWO\ KLJKHU nucleotide substitution rate and may potentially cause endemic outbreak, 5 further study are needed to monitoring the spread of this virus in Indonesia.
Although this serotype was found in this study we are unable to obtain any DENV-4 sequences. To deal with this problem, in the future we could apply methods that utilize shorter fragments of E gene to determine the genotype of our DENV-4.
Laboratory-based dengue surveillance system in Indonesia has just initiated in late 2014 and still ongoing. Prior to that, samples from dengue cases were not collected continuously in sentinel system. Therefore we could not ¿QG FRPSUHKHQVLYH HSLGHPLRORJ\ DQG YLURORJ\ GDWD IURP the related sentinel with consecutive years, the authors aware that many studies and incidental survey concerning dengue were carried out previously. Essential data was generated and dengue sequence could be obtained from public domain such as Genbank. However, continuous surveillance would be useful to understand the dynamic of dengue disease in Indonesia. Even though, some of the information about DENV genotype in Indonesia still can be found in some publication and also in Gene Bank.
In conclusion, we have presented the distribution of DENV genotype in dengue sentinel surveillance sites. These viruses were belongs to various genotypes that have been circulating previously in Indonesia.