A total of 52 subjects, including 15 males and 37 females between 26

and 60 year of age, were enrolled. Of 52 samples, 5 (9.62%) were positive

for dengue infection (Fig. 1A). All dengue-positive samples serotyped

using techniques described previously. Samples S27 and S36 had double-

infections with DENV-1 and DENV-4, and DENV-3 and DENV-4,

respectively (Fig. 1B, Table 1). Single DENV-4 infections were found in

samples S34, S43, and S51. The nucleotide sequences were deposited in

GenBank with the following accession numbers: KM003963 (DENV-1)

and KM003995-KM003998 and KM004003 (DENV-4). Since the DENV-3

nucleotide sequences were less than 200 bp in length, they were not

accepted for deposit in the NCBI system.

Analysis of DENV-1 and DENV-4 serotypes, evolutionary

relationships based on the phylogenetic tree showed that the virus

belonged to the sylvatic genotype (Fig. 2) and genotype II (Fig. 4),

respectively. Interestingly, the DENV-3 tree showed that the virus

belonged to genotypes I and II (Fig. 3).

A B

RESULTS

Double dengue serotypes in asymptomatic populations living

in an area of Thailand endemic for dengue hemorrhagic fever

Kittichai V1, Sriprapun M2, Tawatsin A3, Thavara U3, Siriyasatien P4,5*

1Medical Science Program, Faculty of Medicine, Chulalongkorn University, Bangkok, 2Department of Clinical

Microbiology, Faculty of Medical Technology, Huachiew Chalermprakiet University, Bangkok, 3National Institute of

Health, Department of Medical Sciences, Ministry of Public Health, Nonthaburi, 4Department of Parasitology,

Faculty of Medicine, Chulalongkorn University,Bangkok, 5Excellence Center for Emerging Infectious Disease, King Chulalongkorn Memorial Hospital, Thai Red Cross Society, Bangkok, Thailand

Dengue virus infection remains a major health problem worldwide. Understanding dengue infections and

characterizing circulating viruses are essential for disease prevention and control as well as vaccine development. In this

study, we aimed to identify dengue virus in healthy people living in area endemic for dengue disease. Blood samples were

collected from 52 healthy local subjects living in a dengue-endemic area of Thailand. Viral RNA was detected using

nested reverse transcription polymerase chain (RT-PCR) that amplified the E gene. Phylogenetic trees were constructed

by neighbor-joining method using MEGA6.06. Dengue virus was detected in 5 of 52 samples (9.62%). Double virus

serotypes were identified in two samples. Sylvatic dengue serotype 1 genotypes were also observed. Serotype 4 belonged

to genotype II. Interestingly, both genotypes I and II were found in one serotype 3 sample. These preliminary results may

provide better understanding of dengue infections and viral transmission between populations and mosquitoes. Furthermore, these findings may be significant for epidemiological studies of dengue hemorrhagic fever.

ABSTRACT

Dengue viruses (DENV-1 to DENV-4) are transmitted to human hosts

through the bites of infected female Aedes aegypti and Ae. albopictus

mosquitoes [1]. The disease is problematic worldwide, especially in tropical

and subtropical areas. Of 390 million infected patients reported annually,

50-100 million dengue cases (10-15%) are symptomatic, approximately

500,000 develop DHF [2], and up to 300 million are asymptomatic or mild

cases [3]. However, the relationship between asymptomatic and symptomatic

infections and the role asymptomatic infections play in disease transmission

remains unknown. Based on historical dengue incidence data during 2007

and 2012 obtained from the Department of Disease Control in Thailand’s

Ministry of Public Health, provinces in the central region, such as Samut

Sakhon, are at high risk for dengue outbreaks [4]. therefore, this study tested

for presence of the virus in local adult volunteers with no reported dengue

signs, symptoms, or diagnoses. These findings could provide a better

understanding of dengue pathogenesis and the relationship between the virus

and its hosts in the mosquito-human-mosquito cycle within endemic areas.

This information could be of interest to epidemiological studies and inform development of effective dengue control measures.

INTRODUCTION

1. Thavara, U., Siriyasatien, P., Tawatsin, A., Asavadachanukorn, P., Anantapreecha, S., Wongwanich, R. and Mulla, M.S. 2006. Double infection of heteroserotypes of

dengue viruses in field populations of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) and serological features of dengue viruses found in patients in southern

Thailand. The Southeast Asian Journal of Tropical Medicine and Public Health. 37(3):468-76.

2. Guzman, M.G., Halstead, S.B., Artsob, H., Buchy, P., Farrar, J., Gubler, D.J., Hunsperger E,Kroeger, A., Margolis, H.S., Martínez, E., Nathan, M.B., Pelegrino, J.L.,

Simmons, C., Yoksan, S. and Peeling, R.W. 2010. Dengue: a continuing global threat. Nature Reviews Microbiology. 12 (Suppl 8): S7-S16.

3. Bhatt, S., Gething, P.W., Brady, O.J., Messina, J.P., Farlow, A.W., Moyes, C.L., Drake, J.M., Brownstein, J.S., Hoen, A.G., Sankoh, O., Myers, M.F., George, D.B.,

Jaenisch, T., Wint, G.R., Simmons, C.P., Scott, T.W., Farrar, J.J. and Hay, S.I. 2013. The global distribution and burden of dengue. Nature. 496(7446): 504-507.

4. Suangtho, P., Kaewnorkkao, W. and Sayumpurujinun, S. 2013. 506 Surveillance weekly summarized reports of dengue virus infection during 1 January to 18 April

2013. Bureau of Epidemiology,Department of Disease Control,Ministry of Public Health, Thailand. Available:http://203.157.15.4/wesr/file/y56/F56151_1353.pdf.

Accessed 25 May 2014.

5. Yenchitsomanus, P.T., Sricharoen, P., Jaruthasana, I., Pattanakitsakul, S.N., Nitayaphan, S., Mongkolsapaya, J. and Malasit, P. 1996. Rapid detection and identification of dengue viruses by polymerase chain reaction (PCR). The Southeast Asian Journal of Tropical Medicine and Public Health. 27(2): 228-236.

REFERENCES

We identified asymptomatic dengue virus infections with both single

and co-infections in a highly endemic area of Thailand. Study of

subclinical infections could provide better understanding of disease

pathogenesis in the human host. Moreover, basic knowledge of co-infection

and concurrent transmission in endemic areas are key factors for

epidemiological design of effective control programs. Additionally, data on

variations in viral sequences could be used to support future development of diagnostic and treatment approaches as well as vaccine design.

CONCLUSIONS

This study was supported by His Majesty King Bhumibhol

Adulyadej’s 72nd Birthday Anniversary Scholarship, Graduate School,

Chulalongkorn University; the Graduate School, Chulalongkorn

University and the RatchadapisakSompotch Fund, Faculty of Medicine,

Chulalongkorn University (RA 57/099); and National Science and Technology Development Agency (Thailand) for Research Chair Grant.

ACKNOWLEDGEMENTS

MATERIALS AND METHODS Sample collection

(Blood samples )

RNA extraction

Nested RT-PCR of the dengue

virus envelope (E) gene

DNA Cloning and Sequencing

Phylogenetic tree construction

Adults between 20-60 years of age living in Ban Phaeo

district in the Samut Sakhon province with no history of

dengue infection in the previous year were enrolled in the

study.

The dengue-specific primer sets produced 504, 346, 198, and 143 bp amplicons for serotypes DENV 1-4, respectively [5].

Figure 1 Dengue virus detection by nested RT-PCR amplification of a region of the E-gene. A) Plasma samples positive for dengue virus. M: 100 bp

ladder; P: positive; S27: sample 27; S34: sample 34; S36: sample 36; S43: sample 43, S51: sample 51; N: negative control. B) Typing of dengue viruses

from plasma samples. M: ladder, PD1-4: positive for DENV1-4 respectively; S27-1 to 4: typing of DENV1-4 respectively in sample 27; S36-1 to 4:

typing of DENV1-4 respectively in sample 36; S43-1 to 4: typing of DENV1-4 respectively in sample 43; N: negative control.

Figure 2 A phylogenetic tree of DENV-1 based

on partial E gene sequences. The tree was

constructed using 26 DENV-1 sequences,

including 25 references and one sequence from

study participants (black solid- diamond square

triangle).

Figure 3 A phylogenetic tree of DENV-3 based on

representative partial sequences of the dengue

virus E gene. The tree was constructed with 36

DENV-3 sequences including 24 reference

sequences and two DENV-3 sample sequences

(black-solid diamonds square triangle and black-

solid circular), which were belonged to the

genotype I and genotype II, respectively.

Figure 4 A phylogenetic tree of DENV-4 based

on partial E gene sequences. The tree was

constructed using 24 DENV-4 sequences were

also used, including 19 reference sequences and

five DENV-4 sample sequences (black solid-

diamond square triangle).

Table 1 Results of RT- and nested RT-PCR amplification of the dengue virus E gene

amplification from plasma samples. Samples were collected from local asymptomatic

populations without history of dengue infection during the study period.