Molecular Surveillance of Dengue Virus in Bangkalan, Madura

Island, Indonesia, 2012-2014

Teguh Hari Sucipto

, Tomohiro Kotaki

, Kris Cahyo Mulyatno

, Siti Churrotin

, Shuhai Ueda

Puspa Wardhani

, Aryati

, Soegeng Soegijanto

, and Masanori Kameoka

Indonesia–Japan Collaborative Research Center for Emerging and Re-emerging Infectious Diseases, Institute of

Tropical Disease, Airlangga University, JI, Mulyorejo, Surabaya 60115, Indonesia

Center for Infectious Diseases, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe,

Hyogo 650-0017, Japan

Keywords: Dengue virus, Bangkalan, Molecular surveillance, Serotype

Abstract: The dengue virus (DENV), which belongs to the family Flaviviridae and the genus Flavivirus was endemic

in tropical or sub-tropical areas and was transmitted to human by Aedes aegypti. Bangkalan is one of the

largest cities in the dengue endemic region in East Java Province, Indonesia. This study, aimed to understand

the dynamic of dengue cases in Bangkalan, were performed a molecular surveillance and serotyping. Dengue

detection and serotyping were observed using Reverse Transcriptase-Polymerase chain reaction (RT-PCR).

A total of 359 samples suspected with dengue infection were collected in Bangkalan Madura Island in 2012,

2013, and 2014, which successfully isolated 17 viruses with positive dengue infection. Serotyping study

revealed the predominance of DENV-1, presented in 9 isolated viruses, DENV-2 presented in 7 isolated

viruses, and DENV-3 presented in 1 isolated virus.

1 INTRODUCTION

Dengue is a kind of infectious disease that was

distributed in the tropical and sub-tropical areas

(Green and Rothman, 2006). Dengue was transmitted

to human by Aedes aegypti. More than 250,000-

500,000 dengue infection cases occurred in the world

every years (Wilder-Smith et al. 2010). Dengue

causes various clinical manifestations, ranging from

dengue fever (DF) to more severe forms of the

disease, such as dengue hemorrhagic fever (DHF) and

dengue shock syndrome (DSS) (Martina et al. 2009).

Four distinct serotypes were reported, DENV-1,

DENV-2, DENV-3, and DENV-4.

Indonesia is one of the largest countries in the

dengue endemic region worldwide (Gubler, 2002).

Indonesia is a vast archipelago country that is

regularly affected by the disease. Dengue occurs in all

34 provinces in the country annually and periodic

major outbreaks occur regularly (Nusa et al. 2014).

Bangkalan is a city located in the Madura island,

Indonesia, located 37.8 Km from Surabaya. Dengue

cases were reported in Bangkalan every year

(Department of Health East Java Province, 2013). In

this study, we performed a molecular surveillance

study to understand the dynamic of dengue cases in

Bangkalan. We reported information about the

prevalence of the dengue disease, as well as the

DENV serotype distribution in the city.

2 EXPERIMENTAL

2.1 Samples Collection

This study was approved by the Ethics Committees of

Airlangga University (Ethics Committee Approval

Number: 24-934/UN3.14/PPd/2013) as well as Kobe

University Graduate School of Medicine (Ethics

Committee Approval Number: 784). A total of 359

selected patients having febrile, grading severity of

dengue infection followed by WHO criteria 2011.

Blood samples were collected from patients,

aseptically processed for the separation of serum and

Sucipto, T., Kotaki, T., Mulyatno, K., Churrotin, S., Ueda, S., Wardhani, P., Aryati, ., Soegijanto, S. and Kameoka, M.

Molecular Surveillance of Dengue Virus in Bangkalan, Madura Island, Indonesia, 2012-2014.

DOI: 10.5220/0009843800002406

In Proceedings of BROMO Conference (BROMO 2018) - Symposium on Natural Product and Biodiversity, page 1

ISBN: 978-989-758-347-6

keep in -20 °C deep freeze at hospital. After 2 week

were transferred to -80 °C deep freezer in Institute of

Tropical Disease (ITD) Universitas Airlangga till

processed futher for virus isolation in Vero cell

(Sucipto et al. 2018).

2.2 Virus Isolation in Vero Cell

Serum specimens diluted with Vero culture medium

(1:10) were inoculated to Vero cell monolayer

(Yamanaka et al. 2011) at 37℃ in a 5% CO

for seven

days. After three blind passages, cells were subjected

to immunostaining with flavivirus group cross-

reactive monoclonal antibody (D1–4G2; American

Type Culture Collection, Manassas, VA) to examine

the presence of viral antigens (Konishi et al. 2010).

Antigen-positive cells were subjected to RNA

extraction by using the QIAamp

viral RNA Mini Kit

(Qiagen, Cat. No. 52906).

2.3 Reverse Transcriptase-Polymerase

Chain Reaction (RT-PCR)

Serotyping using multiplex RT-PCR complied by

Lanciotti et al., 1992 (Lanciotti et al. 1992). Reverse

transcriptase-PCR was performed in a Superscript III

(Invitrogen). The PCR amplification reaction was

performed in r-Taq Polymerase (Toyobo) 0.1 µL. The

PCR program was 94°C for 30 second, 94°C for 1

minutes, 50°C for 1 minutes, 72°C for minutes, 72°C

for 7 minutes.

3 RESULT AND DISCUSSION

A total of 359 samples from suspected dengue

infection cases were collected in Bangkalan, Madura

Island in 2012, 2013, and 2014. RT-PCR

confirmations were performed to detect the presence

of virus on those 359 serum samples and successfully

isolated viruses from 17 positive dengue infections

(Table 1). The result of serotyping identified the

presence of all DENV serotypes in Bangkalan, with

DENV-1 as the predominant serotype, followed by

DENV-2 and also DENV-3. Of the 17 isolated

viruses, serotyping study revealed the predominance

of DENV-1, presented in 9 isolated viruses. DENV-2

was presented 7 isolated viruses. DENV-3 was

presented in 1 isolated virus.

The DENVs were detected very low in the

samples. This is due to the storage of serum in the

hospital is not at -80 °C, causing the virus to be

unstable and broken (Young et al. 2000).

The predominant DENV-1 serotypes in

Bangkalan found in this study, is currently reported

in other cities in East Java Province-Indonesia

including, in Surabaya (Kotaki et al. 2014; Yamanaka

et al. 2011). The previous study suggests the

possibility that a DENV-1 outbreak in Bangkalan,

with 459 dengue cases reported in 2013 by

Department of Health of East Java, is similar to that

observed in Surabaya in the same year (Department

of Health East Java Province, 2013). It may be caused

by travellers from Bangkalan that visit Surabaya for

leisure and Bangkalan is one of the regions in Madura

Island that have been supplying workers for cities

Surabaya. Based on data from the governor decision

of East Java Province states that the pay of workers in

Bangkalan is less than in Surabaya.

In Indonesia, dengue occurred for the first time as

an outbreak in Surabaya in 1968 (Hotta et al. 1970;

Sumarmo, 1987). Dengue fever had spread to all

regions of the province with increasing number of

cities infected. In Jakarta from 1975 until 1978,

dengue cases were reported. DENV-3 was

predominantly presented, followed by DENV-2,

DENV-1, and DENV-4 (Sumarmo et al. 1983). In

2004, there was an outbreak of dengue again in

Jakarta, with DENV-3 being the most predominant

serotype, similar to be outbreak in 1975 until 1978,

followed by DENV-4, DENV-2, and DENV-1

(Suwandono et al. 2005). In 2000 to 2002, it was

reported that dengue cases in Bandung, West Java

Province, in which DENV-2 was the predominant

serotype identified (Porter et al. 2005). In Sukabumi,

West Java Province, serotyping revealed the

predominant of DENV-2, followed by DENV-1, and

also DENV-4. DENV-3 was not detected (Nusa et al.

2014). In the Central Java Province, Semarang city,

serotyping identified the presence of all DENV

serotypes, which DENV-1 genotype I and II as the

predominant serotypes, followed by DENV-2 with

cosmopolitan genotype, and also DENV-3 with

genotype I (Fahri et al. 2013). In Surabaya East Java

Province, DENV-1 was reported as predominant in

November 2008 until June 2013, while DENV-2 was

predominantly reported before November 2008 and

after June 2013 (Kotaki et al. 2014; Yamanaka et al,

2011).

Table 1. The result of serotyping study in 17 positive

dengue-infected samples from 2012 to 2014

BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity

No.

Code of

Samples

Serotype

Years

M43

DENV-1

2012

M48

DENV-1

M50

DENV-1

M54

DENV-1

M56

DENV-1

M60

DENV-1

M105

DENV-2

2013

M107

DENV-2

M145

DENV-2

10.

M152

DENV-2

11.

M158

DENV-2

12.

M163

DENV-3

13.

M280

DENV-1

2014

14.

M282

DENV-2

15.

M298

DENV-1

16.

M340

DENV-2

17.

M348

DENV-1

4 CONCLUSIONS

This study has observed the first molecular data of

DENV in Bangkalan, Madura Island, Indonesia.

Serotype predominance is similar to that from

molecular surveillance study in Surabaya, suggesting

the endemic nature of the infecting virus that caused

a high prevalence of dengue in Bangkalan. This study

shows the importance of continuous virus

surveillance in dengue endemic areas better

understanding in the dynamic of dengue infection

disease in Indonesia.

ACKNOWLEDGEMENTS

This work was supported by the joined program of the

Japan Initiative for Global Research Network on

Infectious Disease (J-GRID); Syarifah Ambami Rato

Ebu General Hospital, Bangkalan, Madura Island,

East Java Province, Indonesia; and Institute of

Tropical Disease (ITD) the Center of Excellence

(COE) program by the Ministry of Research and

Technology (RISTEK) Indonesia.

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