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Dengue research doi: 10.1016/S2222-1808(16)61144-1 ©2016 by the Asian Pacific Journal of Tropical Disease. All rights reserved.

Genetic analysis of imported dengue virus strains by Iranian travelers

Nariman Shahhosseini1*, Sadegh Chinikar2*, Norbert Nowotny3,4, Anthony R. Fooks5,6, Jonas Schmidt-Chanasit1

1Bernhard Nocht Institute for Tropical Medicine, WHO Collaborating Centre for Arbovirus and Haemorrhagic Fever Reference and Research, Department

of Virology, Hamburg, Germany

2Pasteur Institute of Iran, Tehran, Iran

3Institute of Virology, Department of Pathobiology, University of Veterinary Medicine, Vienna, Austria

4Department of Basic Medical Sciences, College of Medicine, Mohammed Bin Rashid University of Medicine and Health Sciences, Dubai Healthcare City,

Dubai, United Arab Emirates

5Wildlife Zoonoses and Vector-borne Diseases Research Group, Animal and Plant Health Agency, Woodham Lane, New Haw, Surrey, KT15 3NB, UK

6Department of Clinical Infection, Microbiology and Immunology, University of Liverpool, Liverpool, UK

Asian Pac J Trop Dis 2016; 6(11): 850-853

Asian Pacific Journal of Tropical Disease

journal homepage: www.elsevier.com/locate/apjtd

*Corresponding authors: Nariman Shahhosseini, Department of Virology, Bernhard Nocht Institute for Tropical Medicine, World Health Organization Collaborating Centre for Arbovirus and Haemorrhagic Fever Reference and Research, Hamburg, Germany. Tel: +49 40 42818471 E-mail: shahhosseini@bnitm.de Sadegh Chinikar, Pasteur Institute of Iran, 69 Pasteur Ave., Tehran, Iran Tel: +989351898070 E-mail: sadeghchinikar@yahoo.com The journal implements double-blind peer review practiced by specially invited international editorial board members.

1. Introduction

Dengue virus (DENV), an arthropod-borne RNA virus of the

Flaviviridae family, has 4 serotypes that cause dengue fever

(DF) or dengue hemorrhagic fever (DHF) in humans[1]. The

genome length of DENV is approximately 11 kb and encodes

three structural proteins including capsid, premembrane/

memberane and envelope, and seven nonstructural proteins

(NS1, NS-2A, NS-2B, NS-3, NS-4A, NS-4B and NS-5)[1,2].

DENV consists of four closely related but genetically distinct

virus serotypes (DENV-1 to 4). Phylogenetic studies of

DENV have revealed genetic diversity within each serotype.

DENV-1 has been sub-divided into five genotypes (I–V) based

on phylogenetic analysis of the envelope gene, while other

ARTICLE INFO ABSTRACT

Dengue virus sequences used in this study were obtained from two Iranian patients who were both with a history of traveling to Malaysia. The maximum likelihood phylogenetic tree demonstrated that two sequences were grouped into dengue virus 1. Specifically, strains Iran-DF1 and Iran-DF2 clustered in genotype I and III, respectively.

Contents lists available at ScienceDirect

Article history:Received 1 Sep 2016Received in revised form 18 Sep 2016Accepted 20 Sep 2016Available online 26 Sep 2016

Keywords:Dengue virusPhylogenySerotypeGenotypeEpidemiologyIran

Nariman Shahhosseini et al./Asian Pac J Trop Dis 2016; 6(11): 850-853 851

phylogenetic studies on capsid, premembrane and memberane

genes of DENV-1 have revealed three distinct genotypes

(genotypes I–III)[3,4]. DF is characterized by general signs and

symptoms that could occur with many other illnesses including

general fever, nausea, vomiting, abdominal pain, vomiting and

severe headache. This virus is principally transmitted by Aedes

aegypti mosquitoes[5].

The first report of DF in Iran was in 2008. The patient

had previously travelled to Malaysia (Kuala Lumpur) with a

history of entering a forest[6]. Since 2008, additional human

cases with DF symptoms were diagnosed in Iran. Commonly,

those patients had travelled to Malaysia. Due to the continued

monitoring of DF, 24 cases have now been confirmed in Iran.

The majority of these patients are travelers with a travel

history to Malaysia, India and Thailand[7]. However, DENV

RNA was not detected in these imported DF cases. This

study characterized imported DENV strains in Iran by using

a phylogenetic approach. These data will lead to a better

understanding of DENV epidemiology in Iran and may help to

discriminate imported DENV strains from locally circulating

DENV strains.

2. Material and methods

Two previously healthy women in her 40s and 50s were

treated in a hospital in Iran in 2009 and 2011 after returning

from Malaysia. For the previous days, they had been suffering

from fever (up to 40 °C) and myalgia. Serum samples collected

during the first week of illness gave positive results in DENV

immunoglobulin M ELISA, as well as for DENV RNA with

a DENV-specific RT-PCR, demonstrating an acute DENV

infection[1]. The amplified fragments from the RT-PCR were

purified for Sanger sequenced. The sequences described

in this study have been deposited in the GenBank database

under accession numbers KM669157 (strain: Iran-DF1) and

KP144198 (strain: Iran-DF2).

In addition to the two DENV sequences obtained from Iranian

patients, several sequences representing all serotypes (DENV-1

to 4) and three genotypes within serotype 1 based on capsid,

premembrane and memberane genes (available from GenBank

at www.ncbi.nih.gov) were incorporated into the alignments for

phylogenetic analyses. The sequence alignment was undertaken

using ClustalW and phylogenetic trees were generated by the

maximum likelihood method with kimura 2-parameter distance

using molecular evolutionary genetics analysis software.

Afterwards, analysis of split decomposition was performed

by the Splits Tree 4.0 software to assess the presence of a

“phylogenetic network” at the genotype level for DENV-1[8].

3. Results and discussion

The phylogenetic tree based on maximum likelihood showed

that two sequences obtained from two Iranian patients were

grouped within the DENV-1 lineage. The Iran-DF1 (KM669157)

sequence showed 72% closeness to the 428 bp region, which

corresponds to nt 198–628 in the late region of the capsid

gene and the early region of the membrane gene of the strain

isolated from China (JQ048541). Iran-DF2 (KP144198)

sequence showed 52% closeness to the 428 bp region of the

strain isolated from India (JF815210) (Figure 1).

The phylogenetic network revealed clustering of isolates

in three distinct genotypes (I, II and III). Iran-DF1 clustered

in genotype I with the other viral isolates from Malaysia,

Cambodia, Singapore, Thailand and China. Genotype III

showed three distinct sub-lineages. All Myanmar isolates were

grouped together with significant identity, whilst all isolates

from South America formed a separate sub-lineage. Iran-DF2

formed the third sub-lineage within genotype III with other

isolates from Southeast Asia (Figure 2).

The estimate of dengue patients among the Iranian population

is of concern and is currently being more regularly monitored

by the Iranian health authorities. We performed the first

phylogenetic analysis of imported DENV strains in Iran in order

to trace the potential origin of these strains.

Our data are in accordance with previous reports that

DENV-1 has been sub-divided into three genotypes (I, II and

III) based on phylogenetic analysis of the capsid, premembrane

and memberane genes[3,4]. Recent data present that DENV-1

genotype I and III strains were imported to Iran by travelers

returning from Malaysia. DENV-1 strains are responsible for

the autochthonous DF cases in mainland Europe and thus may

be able to cause autochthonous DF cases in Iran. The main

DENV vectors Aedes aegypti and Aedes albopictus are present

in Iran[9]. Thus, this might increase the risk of the introduction

and circulation of DENV in Iran. We reported the genetic

diversity of DENV strains among Iranian DF patients who

acquired the infection following the travel to DENV-endemic

regions in Southeast Asia. The Iranian public health authorities

should implement preventive measures in order to reduce the

risk of autochthonous DF cases in Iran caused by imported

DENV initially seeded into the local mosquito population by

Iranian travelers.

Nariman Shahhosseini et al./Asian Pac J Trop Dis 2016; 6(11): 850-853852

Conflict of interest statement

We declare that we have no conflict of interest.

Acknowledgments

We thank all of the members of the Arboviruses and Viral Hemorrhagic Fevers Laboratory (National Reference

DENV-1/IR/Iran-DF2/2011 KP14419852

53

7795

92

87

98

89

73

81

95

80

82

72

91

72

73

78

100

100

DENV-4

DENV-2

DENV-3

DENV-1

100

100

DENV-1/IN/Delhi-51/2010 JF815210

DENV-1/BU/DS06-210505/2005 EU179860

DENV-1/SG/8114/1993 AY762084

DENV-1/IN/GWL-19/2002 EU626490

DENV-1/MM/40553/1971 AY713473

DENV-1/MM/40568/1976 AY722801

DENV-1/MM/32514/1998 AY722803

DENV-1/MM/23819/1996 AY722802

DENV-1/VE/BID-V2264/2006 FJ639824

DENV-1/BR/SB 01057805-DF02/2001 AB519681

DENV-1/PY/259par00/2003 AF514883

DENV-1/MY/P72-1244/1972 EF457905

DENV-1/USA/Haw03663/2001 DQ672564

DENV-1/CN/GD95/1995 FJ196846

DENV-1/MY/DH-S1-05-04/2005 JN697056

DENV-1/ID/98901518/1998 AB189120

DENV-1/IN/1412/1998 AY584594

DENV-1/TH/ThD1 048894/1994 AY732475

DENV-1/TH/ThD1-0097-94/1994 AY732480

DENV-1/KH/L0904278/2001 AF538024

DENV-1/TH/ThD1 010201/2001 AY732479

DENV-1/MY/DH-S1-05-152/2005 JN697058

DENV-1/MY/DH-S1-05-154/2005 JN697057

DENV-1/SG/06K2290DK1/2006 EU081281

DENV-1/IR/Iran-DF1/2009 KM669157

DENV-1/CN/DG14/2011 JQ048541

DENV-3/IN/ND143/2007 FJ644564

DENV-3/LK/IMTSSA-SRI/2000 AY099336

DENV-3/TW/99TW628/1999 DQ675533

DENV-2/TW/BID-V5056/2008 HQ891024

DENV-2/ID/1022DN/1975 GQ398268

DENV-2/TH/ThD2-049884/1984 DQ181804

DENV-4/VE/BID-V2173/1999 FJ639745

DENV-4/CO/BID-V3409/2001 GQ868582

DENV-4/CO/341750/1982 GU289913

DENV-4/BR/H778887/2011 JQ513337

0.05

97

Figure 1. The maximum likelihood phylogenetic tree of DENV serotypes constructed by using partial nucleotide sequence of the capsid, premembrane

gene (length, 511 nucleotides) of DENV strains.

For each sequence used, the standardized DENV strain nomenclature and GenBank accession number are shown. The DENV strains from this study are in

bold.

Nariman Shahhosseini et al./Asian Pac J Trop Dis 2016; 6(11): 850-853 853

Laboratory), Pasteur Institute of Iran. This study was conducted

in the framework of national surveillance system on arboviruses

in Iran, and all human probable samples for arboviruses and

viral hemorrhagic fevers were sent for screening.

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Figure 2. Phylogenetic analysis of the neighbor-net network of DENV-1 genotypes constructed by using partial nucleotide sequence of the capsid, premembrane gene (length, 511 nucleotides) of DENV strains. For each sequence used, the standardized DENV strain nomenclature and GenBank accession number are shown. The DENV strains from this study are in bold.

MY/EF457905

MM/AY722802

MM/AY722803

MM/AY713473

MM/AY722801

BR/AB519681

PY/AF514883

VE/FJ639824

SG/AY762084

IN/EU626490

BU/EU179860 USA/DQ672564MY/JN697056

ID/AB189120

IN/AY584594

TH/AY732480TH/AY732475

MY/JN697058MY/JN697057

SG/EU081281CN/JQ048541

TH/AY732479

KH/AF538024 IR/KM669157

Genotype I

CN/FJ196846

Genotype II

IN/JF815210

IR/KP144198

SylvaticGenotype III