Medical

Communication

Biosci. Biotech. Res. Comm. 10(1): 11-21 (2017)

Serotypes of dengue viruses circulating in Jazan

region, Saudi Arabia

A.A. Alsheikh

, O.M. Daffalla

, E.M. Noureldin

, W.S. Mohammed

, K.J. Shrwani

Y.A. Hobani

, A.D. Abkar

, F.A. Alsheikh

and A.M. Assiri

National Center for Vector-Borne Diseases, MoH-Jazan, Saudi Arabia

Department of Medical Parasitology, Faculty of Medical Laboratory Sciences, University of Gezira, Sudan

Faculty of Public Health and Tropical Medicine, Jazan University, Saudi Arabia

Minister Deputy Assistant for Preventive Health, MoH-Riyadh, Saudi Arabia

ABSTRACT

Dengue fever is considered to be the most important mosquito-borne disease and considered as endemic disease in

Jazan region, Saudi Arabia. The present study aimed to analyze the prevailing dengue virus serotypes for the  rst

time in the region. Serum samples of 220 suspected dengue cases were collected throughout 2016 and tested by

one step Reverse Transcription Polymerase Chain Reaction (RT-PCR) with a set of speci c primers for detection of

four dengue virus serotypes followed by sequencing the PCR products to con rm the results. Out of the 220 serum

samples, 124 were found positive for dengue infection (56.4%). Three dengue virus serotypes were detected; DEN-1,

DEN-2 and DEN-3. DEN-2 is the most common and predominant type in the region rating 83.9% (104/124), followed

by DEN-1 8.9% (11/124), and then DEN-3 7.2% (9/124). The high seroprevalence of dengue virus infections in Jazan

region indicates its endemicity. The present study highlights the importance of tracking the spread of dengue virus

types and its implication for analyzing changes in dengue endemicity in speci ed areas over time. Complete genome

sequencing is required for the three detected dengue virus serotypes circulating in the region (DEN-1, DEN-2, and

DEN-3) to serve as references for any future epidemiological researches and/or outbreaks.

KEY WORDS: DENGUE FEVER, SEROTYPES, 1, 2, AND 3, JAZAN REGION, SAUDI ARABIA

ARTICLE INFORMATION:

*Corresponding Author: adelalsheikh@gmail.com

Received 1

Feb, 2017

Accepted after revision 26

March, 2017

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Online ISSN: 2321-4007 CODEN: USA BBRCBA

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Online Contents Available at: http//www.bbrc.in/

12 SEROTYPES OF DENGUE VIRUSES CIRCULATING IN JAZAN REGION, SAUDI ARABIA BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Alsheikh et al.

INTRODUCTION

Dengue fever is considered to be the most important

re-emerging vector-borne disease worldwide and is

endemic in more than 125 countries (Murray et al.,

2013). Four hundred million of cases are estimated to

occur annually (CDC, 2016). Dengue is a viral disease

transmitted to humans by the bite of infected females of

the main vector Aedes aegypti and to a lesser extent by

Aedes albopictus mosquitoes (WHO, 2009).

There are  ve genetically related but antigenically

distinct single-stranded RNA serotypes belonging to

Flaviviridae family and genus F lavivirus; DEN-1, DEN-

2, DEN-3, DEN-4 (WHO, 1997), and DEN-5 (Mustafa

et al., 2015).

However, no cross protection occurred between the

dengue serotypes, the immunity is serotype speci c.

According to disease severity, the World Health

Organization has classi ed dengue into three categories;

Dengue Fever (DF), Dengue Hemorrhagic Fever (DHF),

and Dengue Shock Syndrome (DSS) (WHO, 1997).

Unplanned urbanization and climatic factors, includ-

ing high temperatures and rainfall, might contribute to

epidemics of dengue (Mackenzie et al., 2004; Crowell

et al., 2011; Banu et al., 2011).Aedes mosquito is found

in the urban settings, especially in tropical areas, where

it maintains a sustainable relationship with humans

leading to reemergence of dengue infections and creat-

ing a public health threat (Glenn and Sia, 2008).Spa-

tial patterns in the recent and sequential circulation of

DEN1-5, along with the host and virus genetics, should

be regarded as potential population risk factors for sever

forms of dengue fever (Guilarde et al., 2008; Chaturvedi

2006) because most secondary infection bearing heter-

ologous dengue virus type may lead to severe disease

complications (Green and Rothman, 2006; Vaughn et al.,

2000; Gibbons and Vaughn, 2002; Rico-Hesse, 2003).

In Saudi Arabia, the  rst dengue outbreak has been

reported in 1994 in Jeddah with 289 con rmed cases,

and DEN-1 and DEN-2 were circulating dengue virus

serotypes (Fakeeh and Zaki, 2001). Since then, several

dengue fever outbreaks have been recorded in Saudi

Arabia (Fakeeh and Zaki, 2003; Ahmed, 2010; Khan et

al, 2008; Ayyub et al., 2006; El-Badry et al., 2013; Zaki

et al., 2008) and Yemen (Madani et al., 2013). The case

fatality rate was 4.6 per thousand in 2007 (Saudi Min-

istry of health, 2007).The incidence of dengue fever has

increased in Saudi Arabia during the past few years;

6512 cases in 2013; 2081 cases in 2014; and 4312 cases

in 2015 (Saudi Ministry of health, 2016).

The emergence of DEN-3 in Jeddah was in 1997

(Fakeeh and Zaki, 2001), and since then all the 3 dengue

serotypes (DEN 1–3) were being circulated in the city

(Azhar et al., 2015). Recently, Organji et al (2017) have

reported DEN-1, DEN-2, and DEN-3 to be circulated in

Makkah city. In Jazan region, there were 1790 con rmed

dengue cases between 2005 and 2016 with highest out-

breaks in 2016 (555 cases), followed by 2010 (290 cases),

and 2012 (289 cases) (Dengue control program in Jazan).

Al-Arzaqi et al (2013) reported dengue prevalence of up

to 26.5% in Jazan region, while Gamil et al (2014) noted

47.74% dengue positivity rate in the area.To the best of

our knowledge, no data has been published on the cir-

culation of dengue virus serotypes in Jazan region, thus

our present study is the  rst of its own and aimed to

analyze the prevalence of dengue virus serotypes circu-

lating in Jazan region, southwest of Saudi Arabia.

MATERIAL AND METHODS

Study area: Jazan Region in Southwest Saudi Arabia lies

between 16°-12, and 18°-25, latitude north. It is bordered

in the South by Arabic republic of Yemen with total area

of about 22,000 km² and 1.3 million populations (cen-

sus 2011). Thirty percent of the population concentrated

in six major cities, and the remainders living in over

3500 villages (Alsheikh, 2011). Jazan region is situated

in the subtropical zone and has average monthly tem-

peratures ranging between 25.8°C in January to 33.4°C

in July. The average relative humidity ranges between

55% and 72.5%. The rainy season is started at August

through October with a monthly average of 77 and 56.7

mm, respectively (Alsheikh, 2011). Jazan is divided into

eleven small Governates (Al-Aridah, Damad, Twal, Al-

Ahad, Jazan , Al-Khobah, Samttah, Abuareesh, Sabyah,

Beash and Al-Darb), these locations (Fig.1) although

with different altitudes and geographical Characteristics,

they are almost share the same demographical, agricul-

tural, educational, cultural, housing, health system, and

environmental characteristics.

Sampling: During 2016 about 220 suspected dengue

fever patients serum samples included in this study were

collected from  ve different hospitals in Jazan region

and stored at -80° till further use.

RNA isolation: High Pure Viral Nucleic Acid Kit

from Roche applied science (Germany) used for extrac-

tion of RNA follow the manufacture procedure; 200 µl

of binding buffer supplemented with poly (A) and 50

µl Proteinase K added to 200 µl of serum sample then

mixed immediately and incubated for 10 minutes at

72°C. Addition of 100 µl Proteinase K was mixed with

sample and transferred to High Filter Tube inserted into

Collection Tube. After centrifugation for 1 minute at

10000 rpm, the collection tube was discarded. The  l-

ter tube combined with new collection tube and 500 µl

of inhibitor removal buffer was added and centrifuged

for 1 minute at 10000 rpm. After changing collection

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS SEROTYPES OF DENGUE VIRUSES CIRCULATING IN JAZAN REGION, SAUDI ARABIA 13

Alsheikh et al.

tube, the high  lter tube washed twice by adding 450

µl of wash buffer at the same condition of centrifuga-

tion, followed by centrifugation for 15 seconds at 13000

rpm to remove any residual wash buffer. Then the high

 lter tube was inserted into nuclease free, sterile 1.5 ml

centrifuge tube and 50 µl of elution buffer was added to

elute the viral nucleic acid by centrifugation at 10000

rpm for 1 minute.

Reverse Transcriptase Polymerase Chain

Reaction (RT-PCR)

One step RT- PCR is a rapid, sensitive, and simple for

dengue serotype-speci c diagnosis method. The test was

performed according to the protocol of Lanciotti et al

(1992) with some modi cation; DEN consensus prim-

ers and serotype-speci c primers (Table 1) were used to

amplify the viral genome in this study and synthesized

in Integrated DNA Technology (Belgium). The one step

RT-PCR reactions were performed according to access

RT-PCR–system protocol (Promega-USA) in total vol-

ume of 50 µl containing 10 µl of AMV/T 5X Reaction

Buffer, 1 µl of dNTP Mix (10mM each dNTP,  nal con-

centration 0.2mM), 2 µl of 25mM MgSO4 ( nal concen-

tration 1mM), 1 µl of AMV Reverse Transcriptase 5u/

l ( nal concentration 0.1u/l), 1 µl of T DNA Poly-

merase 5u/l ( nal concentration 0.1u/l), 50pmol ( nal

concentration 1M) of each forward (D1) and reverse

(D2) primers, 5 µl of RNA virus and nuclease free water

to total volume 50 µl. The thermal cycling incubations

temperatures programmed as follows: incubation for 1

hour at 42°C (to convert the RNA to cDNA) then initial

denaturation for 3 minutes at 94°C followed by 35 cycle

of denaturation (94°C, for 30 second), primers annealing

(55°C for 1 minute), primer extension (72°C for 2 min-

utes) and  nal extension for 5 minutes.

Nested-PCR

Nested PCR was performed in 2 tubes for each sample

in 50 l reaction mixture containing 25 µl GoTag®G2

green master mix ready to use from Promega, 10 µl of

the diluted (1:100) RT-PCR product, 50 pmol ( nal con-

centration 1M) of each forward primer D1 and TS1,

TS3 as reverse primers for the  rst tube and TS2, TS4

as reveres primers for another tube. The samples were

subjected to initial denaturation at 94°C for 3 minutes,

30 cycles of denaturation (94°C, 30 s), primer annealing

(55°C, 1 min), primer extension (72°C, 2 min) and  nal

extension for 5 minutes. In each run negative and posi-

tive controls were included. The PCR products of nested

ampli cation were analyzed by gel electrophoresis (1.5

agarose in Tris-Acetate EDTA buffer) staining with eth-

idium promide. The visualization was carried out using

Gel Doc XR Imaging System (Bio-Rad).

Sequencing and bioinformatics analysis

Puri cation and standard sequencing for RT- PCR prod-

ucts were performed by Macrogen Company (Seoul,

Korea). Sequencing reactions were performed in a MJ

Research PTC-225 Peltier Thermal Cycler using a ABI

PRISM® BigDyeTM Terminator Cycle Sequencing Kits

with AmpliTaq® DNA polymerase (FS enzyme) (Applied

Biosystems), following the protocols supplied by the

manufacturer. Single-pass sequencing was performed

on each template using D1 (forward) primer. The

 uorescent-labeled fragments were puri ed from the

unincorporated terminators with Big Dye®X Terminator™

puri cation protocol.The samples were resuspended in

distilled water and subjected to electrophoresis in an ABI

3730xl sequencer (Applied Biosystems). The sequences

were searched for sequence similarity through BLAST

FIGURE 1. Map of Jazan region showing the different Governates.

Alsheikh et al.

14 SEROTYPES OF DENGUE VIRUSES CIRCULATING IN JAZAN REGION, SAUDI ARABIA BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

(www.ncbi.nlm.nih.gov/BLAST/) (Atschul et al., 1997)

and compared to reference sequences of Dengue sero-

types detected in BLAST and downloaded from GenBank

(www.ncbi.nlm.nih.gov/genbank/).

Similarity tree was obtained from database online by

phylogeny.fr (http://www.phylogeny.fr/).

RESULTS

RT-PCR and Nested-PCR: One hundred twenty four

samples out of 220 (56.4%) suspected patient serum

samples tested by RT-PCR were con rmed positive for

dengue virus when using D1 and D2 primers (511bp)

for all serotypes, and the RT-PCR product was used as

a sample for the nested-PCR using a set of serotype-

speci c primers pair as described in the methodology.

Three dengue virus types (DEN-1, DEN-2 and DEN-3)

were detected and the results showed that DEN-2 is the

most common and predominant type in Jazan region

rating one hundred four out of one hundred twenty four

(83.9%), followed by DEN-1 (eleven out of one hundred

twenty four, 8.9%), and then DEN-3 (nine of one hun-

dred twenty four, 7.2%) and serotype 4 was not detected

(Table 2 and Fig. 2).

Sequencing: To con rm the serotype-speci c results,

the partial sequencing was done for nineteen RT-PCR

product samples represent the three serotypes (DEN-1,

DEN-2, and DEN-3). The Blast search showed that the

sequences of our samples aligned along with many pub-

lished sequences of dengue virus serotypes as shown in

Table 3 and Fig.3, Fig.4 and Fig.5, and similarity tree

(Fig.6, Fig.7 and Fig.8) which illustrates the Gen bank

accession numbers and the country of isolates.

Table 1. oligonucleotide primers used in RT-PCR and Nested-PCR

primer Sequence 5 - 3

Genome

position

Size in bp

D1 TCAATATGCTGAAACGCGCGAGAAACCG 134-161 511

D2 TTGCACCAACAGTCAATGTCTTCAGGTTC 616-644 511

TS1 CGTCTCAGTGATCCGGGGG 568-586 482 (Dl and TS1)

TS2 CGCCACAAGGGCCATGAACAG 232-252 119 (Dl and TS2)

TS3 TAACATCATCATGAGACAGAGC 400-421 290 (Dl and TS3)

TS4 CTCTGTTGTCTTAAACAAGAGA 506-527 392 (Dl and TS4)

Table 2. Results of dengue virus serotyping using RT-PCR and nested-PCR

No of samples +ve DEN +ve DEN-1 +ve DEN-2 +ve DEN-3 +ve DEN-4

220 124 (56.4%) 11 (8.9%) 104 (83.9% ) 9 (7.2 %) 0 (0%)

FIGURE 2. Agarose gel electrophoresis of RT-PCR (D1, D2 primers) and

nested-PCR by the speci c primers. Lane 1 and 8 DNA 100bp marker,

lane (2) negative control, lane (3) positive sample DEN-1, lane (4,5)

positive samples DEN-2, lane (6) positive sample DEN-3 and lane (7)

positive RT-PCR product sample (D1 and D2 primers for all serotypes).

Alsheikh et al.

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS SEROTYPES OF DENGUE VIRUSES CIRCULATING IN JAZAN REGION, SAUDI ARABIA 15

Sequencing of DEN-1 in this study revealed that it is

in close similarity to some Asian (Taiwan, Saudi Arabia,

Thailand, Vietnam, and Cambodia) and African (Djibouti

and Eritrea) types (Table 3, Fig.3, and Fig.6). DEN-2 on

other hand, is similar to varies Indian types (Table 3,

Fig.4, and Fig.7), while DEN-3 is in similarity to some

Asian types including India, China, and Singapore (Table

3, Fig.5, and Fig.8).

DISCUSSION

Jazan region has witnessed several outbreaks during the

recent decade (290 cases in 2010, 289 cases in 2012, and

555 cases in 2016 – Dengue control program in Jazan).

The current available data on dengue in Jazan has con-

centrated mainly on serological surveys (Al-Arzaqi

et al., 2013; Gamil et al., 2014) and has not analyzed the

circulating serotypes in the region.

Table 3. Results of RT-PCR and nested-PCR

DEN-1 DEN-2 DEN-3

Gen bank

accession No

Country

Gen bank

accession No

Country

Gen bank

accession No

Country

AB608788 Taiwan JN935383 India KM097092 India

KJ649286 Saudi Arabia KU351296 India KM097092 Singapore

JN638338 Thailand GU968539 India KF954949 China

AF298808 Djibouti KX577706 China GQ466079 India

Z74047 Vietnam KT180256 India FJ644564 India

AF538024 Cambodia KU351306 India DQ317393 India

KU509258 Eritrea JQ639472 India KU216208 India

FIGURE 3. Identities between DEN-1 from Jazan and DEN-1 of Tai-

wan (dbj|AB608788.1) Dengue virus 1 gene for polyprotein, com-

plete cds, strain: 832, Length=10693, Score = 778 bits (421), Expect

= 0.0, Identities = 459/477 (96%), Gaps = 4/477 (1%), Strand= Plus/

Plus.

Alsheikh et al.

16 SEROTYPES OF DENGUE VIRUSES CIRCULATING IN JAZAN REGION, SAUDI ARABIA BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Our results indicated that dengue fever is becoming

highly prevalent in Jazan region (56.4 %) compared to

the previous reports of Al-Arzaqi et al (2013) and Gamil

et el (2014) who reported dengue prevalence of 26.5%

and 47.74% , respectively, in the region. In this study,

three dengue virus types (DEN-1,DEN-2 and DEN-3)

were found circulating in Jazan region with the pre-

dominance of DEN-2 scoring 104 out of 124 dengue

FIGURE 4. Identities between DEN-2 from Jazan and DEN-2 of

India(gb|JN935383.1) Dengue virus strain VCRC/DENV2/03/10

polyprotein gene, partial cds Length=508, Score = 848 bits (459),

Expect = 0.0, Identities = 471/476 (99%), Gaps = 3/476 (1%),

Strand=Plus/Plus

FIGURE 5. Identities between DEN-3 from Jazan and DEN-3 of India

(gb|KF954949.1) Dengue virus 3 isolates 13GDZDVS30E, complete

genomeLength=10677 Score = 861 bits (466), Expect = 0.0, Identi-

ties = 473/476 (99%), Gaps = 1/476 (0%, Strand=Plus/Plus

Alsheikh et al.

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS SEROTYPES OF DENGUE VIRUSES CIRCULATING IN JAZAN REGION, SAUDI ARABIA 17

FIGURE 6. DEN-1 serotype similarity tree.

positive samples (83.9%), followed by DEN-1 (11 out of

124 - 8.9%), and then DEN-3 (9 of 124 - 7.2%), how-

ever serotype 4 was not detected in any of the 124 den-

gue cases. This  nding is in complete accordance with

the work of Fakeeh and Zaki (2001) who reported that

DEN-2 was the predominant serotype, followed by DEN-

1, and DEN-3 in Jeddah, Saudi Arabia. Whereas Organji

et al (2017) in Makkah city, showed that DEN-1 was the

predominant dengue virus type, followed by DEN-2 and

then DEN-3, although the positive blood samples they

used were only six.

The results also coincide partially with the  ndings of

Khan et al (2008) who reported high prevalence of the

DEN-2 in contrast to the prevalence of DEN-1 found by

Organji et al (2017) in Makkah city.In Jeddah, Zaki et al

(2008) revealed that DEN-1 and DEN-2 caused the major

outbreak in 1994, while DEN-3 emerged in 1997. More-

over, they indicated two genotypes for DEN-1 (America-

Africa genotype, and Asia-2 genotype), DEN-2 genotype

clustered within Cosmopolitan genotype, and DEN-3

clustered within genotype III.

In the present study, we found DEN-2 to be the pre-

dominant dengue virus type, a result which is in line

with the reports of Fakeeh and Zaki (2001, 2003) and

Zaki et al. (2008) who stated that DENV-2 virus is the

predominant serotype in Saudi Arabia particularly in

western Saudi Arabia since 1992.El-Kafrawy et al. (2016)

showed that DEN-2 isolate from Jeddah belongs to the

Cosmopolitan genotype was most genetically related to

isolates from Pakistan circulating from 2008 to 2013.The

Alsheikh et al.

18 SEROTYPES OF DENGUE VIRUSES CIRCULATING IN JAZAN REGION, SAUDI ARABIA BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

FIGURE 7. DEN-2 serotype similarity tree.

three dengue virus serotypes DEN-1, DEN-2, and DEN-3

are thought to be predominant in the Middle East, espe-

cially in Yemen and Saudi Arabia (Nedjadi et al., 2015).

Dengue viruses circulating locally in Saudi Arabia are

likely to have been imported into Saudi Arabia by Saudi

traveling abroad to dengue endemic countries, or during

Hajj and Umrrah seasons, or by migrant labour (Zaki

et al., 2008).The introduction of the three dengue virus

types in Jazan region may be resulted from several fac-

tors; traveling of the Jazan citizens for Hajj and Umrrah

or for trade or other purposes, or by traveling abroad to

dengue endemic countries, or by migrant labour, or due

to the proximity of Jazan region to Yemen where the

disease is endemic and the three dengue virus serotypes

DEN-1, DEN-2, and DEN-3 are circulating.

The close similarity of DEN-1 in this study to some

Asian (Taiwan, Thailand, Vietnam, and Cambodia) and

African (Djibouti and Eritrea) types, the similarity of

DEN-2 to varies Indian types, in addition to, the similar-

ity of DEN-3 to some Asian types including India, China,

and Singapore suggested the likelihood of introduction

of theses serotypes to Jazan region either by travel-

ling from and to those countries especially the migrant

labours (DEN-1,DEN-2, DEN3,), or through direct intro-

duction from Jeddah (DEN-1 Jeddah genotype) and

Yemen which is closet to Djibouti and Eritrea (DEN-1

African origin).

It is stated that shifts in circulating dengue virus type

or introduction of new dengue virus type in endemic

areas have shown to be related with incidence of severe

Alsheikh et al.

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS SEROTYPES OF DENGUE VIRUSES CIRCULATING IN JAZAN REGION, SAUDI ARABIA 19

FIGURE 8. DEN-3 serotype similarity tree.e

dengue infections; DHF and DSS (Messer et al., 2003;

Rico-Hesse et al., 1997). Moreover, it is worthy to note

that the primary infections by DEN-1 and DEN-3 are

related with more dengue sever infections, whereas

infections with DEN-2 and DEN-4 are associated with

increased dengue severity when they present as second-

ary infections (Balmaseda et al, 2006). Results of our

study have analysed multiple dengue serotypes which

would help in providing clear evidence of current active

dengue transmission and endemicity in Jazan region.

CONCLUSION

The results of this study reported for the  rst time the

dengue virus types DEN-1, DEN-2, and DEN-3 circu-

lating in Jazan region with the DEN-2 being the pre-

dominant one. The high seroprevalence of dengue virus

infection in Jazan region indicates its endemicity. The

present study highlights the importance of tracking the

spread of dengue virus types and its implication for ana-

lyzing changes in dengue endemicity in speci ed areas

over time. Continuous surveillance of dengue virus sero-

types in the region to detect as earlier the local origin

circulating serotypes from the imported ones especially

new types DEN-4 and DEN-5, for which continued sur-

veillance is imperative. Complete genome sequencing is

required for the three detected dengue virus serotypes

circulating in the region (DEN-1, DEN-2, and DEN-3)

to serve as references for any future epidemiological

researches and outbreaks.

Alsheikh et al.

20 SEROTYPES OF DENGUE VIRUSES CIRCULATING IN JAZAN REGION, SAUDI ARABIA BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

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