Molecular Diagnosis of Chikungunya virus (CHIKV) and Dengue virus (DENV) and its concomitant circulation in South Indian population

    Molecular Diagnosis of Chikungunya virus (CHIKV) and Dengue virus (DENV) and its concomitant circulation in South Indian population Seetha Dayakar, Iravathy K. Goud, Heera Pillai, Viji Remadevi, Sanjai Dharmaseelan, Radhakrishnan R. Nair, M. Radhakrishna Pillai PII: DOI: Reference:

S2214-6695(15)00005-7 doi: 10.1016/j.virep.2015.05.001 VIREP 11

To appear in:

Virology Reports

Received date: Revised date: Accepted date:

27 October 2014 13 April 2015 5 May 2015

Please cite this article as: Dayakar, Seetha, Goud, Iravathy K., Pillai, Heera, Remadevi, Viji, Dharmaseelan, Sanjai, Nair, Radhakrishnan R., Pillai, M. Radhakrishna, Molecular Diagnosis of Chikungunya virus (CHIKV) and Dengue virus (DENV) and its concomitant circulation in South Indian population, Virology Reports (2015), doi: 10.1016/j.virep.2015.05.001

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT S. Dayakar et al. / Virology Reports Short Communication

RI

PT

Molecular Diagnosis of Chikungunya virus (CHIKV) and Dengue virus (DENV) and its concomitant circulation in South Indian population

NU

SC

Seetha Dayakara,b, Iravathy K Goudb, Heera Pillaia,Viji Remadevia ,Sanjai Dharmaseelana, Radhakrishnan R Naira*, M Radhakrishna Pillaia

a

MA

Department of Laboratory Medicine and Molecular Diagnostics, Rajiv Gandhi Centre for Biotechnology, Poojappura, Thycaud,Thiruvananthapuram-695014, Kerala, India b

AC CE P

TE

D

Department of Molecular Biology and Cytogenetics, Apollo Health City, Jubilee hills, Hyderabad-500033,Andhra Pradesh, India

*Corresponding to: Department of Laboratory Medicine and Molecular Diagnostics, Rajiv Gandhi Centre for Biotechnology, Poojappura, Thycaud, Thiruvananthapuram-695014, Kerala, INDIA e-mail: [email protected] (Radhakrishnan R. Nair) Ph:+914712529564,Fax:+914712346333

1

ACCEPTED MANUSCRIPT S. Dayakar et al. / Virology Reports

Introduction

PT

Having known for its menace in tropical countries such as Africa and Asia, dengue fever is caused by a class of pathogens called as flavivirus that belongs to the family of flaviviridae and

RI

Chikungunya, being caused by the alphavirus of family Togaviridae (Buchen et al., 2000). Both

SC

diseases are transmitted to humans by day-biting Aedes aegypti and Aedes albopictus mosquitoes

NU

and almost cause similar clinical symptoms such as fever, rashes, joint pain; headache, fatigue, nausea, vomiting, and body pain are the hallmark for both the diseases that makes diagnosis

MA

difficult (Konstantin et al.,2007). Concomitant circulation of both these viruses in humans was reported and apprehended by serological analysis but their results did not pave clue about the on-

D

going infection with both the viruses (Arankalle et al., 2007). Co-infection of these viruses

TE

presents similar clinical signs, but different disease patterns. Unavailability of viral specific

AC CE P

diagnostic tools complicates medical management strategies and hence clinically viable and easily available molecular markers are the need of the hour. Here, a study was conducted to establish a handy diagnosis method for the screening of chikungunya and dengue fever through in house designed RT-PCR approach.

Results 1024 patient blood samples from Kerala and Andhra Pradesh were tested for the screening of Dengue and Chikungunya. In the current cross-sectional study, we found that nearly 46/105 samples (43.8%) were positive for DENV, 34/105 samples (32%) were positive for CHIKV and 24/105 samples (23%) were positive for the presence of both viruses in the Andhra Pradesh region. In contrast to the above figures, patients from Kerala were found to be positive for

2

ACCEPTED MANUSCRIPT S. Dayakar et al. / Virology Reports dengue with 16.1% (148/919), positive for CHIKV at 2.3% (21/919) and surprisingly only 0.1% (1/919) of people were found to be carrying both the viruses. Serology analysis using Dengue

PT

IgM in Andhra Pradesh and Kerala showed 60.9 % (64/105) and18.8 % (173/919), respectively

RI

(Table 3). To further validate our above findings, we ran RT PCR to detect the presence of

SC

DENV RNA in blood samples of both Andhra Pradesh and Kerala Patients. We concluded that, in Andhra Pradesh alone, 45.6% (21/46) of the samples showed positive for dengue serotype-2;

NU

54.6% (25/46) showed positive for more than one dengue type such as dengue type 2 and 3; co infection was detected in 39.1%; 0.6% (4/46) of the samples being positive for type 2 and 4;

MA

mere 6.5%of all patient samples showed presence of D-1, D-1/2, D-1/4, D-1/3, D-3/4, D-1/3/4,

D

D-2/3/4 serotypes (Table 4).

TE

148/919 samples from Kerala were tested for DENV RNA by RT PCR. Five samples could

specific PCR.

AC CE P

not be genotyped due to low copy number and 143 samples were analyzed for Dengue serotype Among 143 samples, dengue serotype 2 occupied 35.6% (51/143) and the

remaining 64.3% (92/143) of the samples were diagnosed with other subtypes of dengue such as D-4 of 16.7% (24/143), D-3 of13.2% (19/143), respectively. Co-infection of D-2 and D-4 was found to be 12.5% (18/143), D-2 and D-3 were 7.6% (11/143) and co infection was 13.9% (20/143) of the samples tested (Table 4). The BLAST data confirmed the amplified products to be that of DENV and CHIKV sequences. The phylogenetic analyses of the DENV sequences revealed that the amplified DENV samples clustered along with DENV-2, DENV-3 and DENV-4 genotypes (Fig.2), while the representative sequenced CHIKV sample clustered with central African genotype (Fig.3), results of RT PCR showed statistical significant DENV alone vs. Co-infection CHIKV and DENV at 59.9% vs.

3

ACCEPTED MANUSCRIPT S. Dayakar et al. / Virology Reports 23.1% (p value-0) and CHIKV alone vs. Co-infection CHIKV and DENV at 34.3% vs. 23.1% (p value <0.001).

PT

Discussion

RI

There have been very few reports on the co-existence of CHIKV and DENV, particularly in

SC

those living in areas endemic to CHIKV and DENV. Infections with CHIKV and DENV present similar clinical symptoms in patients. Accordingly, there have been reports of co-infection with

NU

CHIKV and DENV in human population living in Asian countries. Some of the data on co-

MA

infection is based on the serological diagnosis of the patient samples but it suggests little details on live infection. In this context, we have proven that the in-house designed primers for RT

D

PCR-based tool enabled a diverse yet commendable diagnosis of CHIKV and DENV as well as

TE

co-infections of both the viruses. In our study, we found 23% (24/105) and 0.1% (1/919) of the

AC CE P

samples was positive for both CHIKV as well as DENV in AP and Kerala respectively, suggesting the co-existence of these two viruses in these patients. Although the virulence and viral loads of CHIKV strains isolated during our study is yet to be established, the clinical spectrum of the two infections is very much similar and can co-exist in the same host (Vanlandingham et al., 2005). Our study also reflects a comparative study of clinical features between monotypic and dual infection cases with chikungunya virus and dengue virus in west Bengal, India (Debjani et al., 2012). The sequence obtained from the RT-PCR assay can be used for phylogenetic neighborhood joining tree analysis and genotyping of the isolates. Thus, the assay can also serve as a tool for rapid clustering of isolates for the genotyping of viruses from different outbreaks. RT-PCR can be applied as a potential rapid test to detect dengue and chikungunya viral infections simultaneously in clinical samples along with the determination of DENV serotypes. 4

ACCEPTED MANUSCRIPT S. Dayakar et al. / Virology Reports

Concluding remarks

PT

False negatives are always plausible if diagnostic tests for both the viruses are not performed.

RI

We have shown that it is possible for clinicians to use simple clinical and laboratory variables to predict these infections for more personalized treatment. Though, we have described an easy

SC

technique here, we are unaware of the sensitivity of the test as the former requires periodic

NU

follow up of the patients who had received respective treatments on diagnosis; having said that, we believe this method would improve the diagnosis and differentiation of viral co-infection,

MA

particularly in tropical areas where health care are in resource-limited settings.

TE

D

Materials and methods

AC CE P

To investigate concomitant circulation of the two viruses, 3-4 ml of blood samples were collected with sterile Vaccutainer™ containing K3 anti-coagulant from clinically suspected DENV/CHIKV 1024 patients who were enrolled after their consent obtained, Apollo Hospitals, Hyderabad, Andhra Pradesh (AP) between August, 2011 and December, 2013 and Thiruvananthapuram (during the local outbreak of CHIKV/DENV), Kerala, India, respectively (Yergolkar et al., 2006). The case definition is given in detail in Table 1. The molecular detection of CHIKV and/or DENV was done through RT-PCR with in house designed primer. Sample vials were centrifuged at 1500 rpm for 10 minutes and plasma/serum was immediately stored at 20⁰C for further analysis. The samples were collected as a part of the outbreak investigation.

5

ACCEPTED MANUSCRIPT S. Dayakar et al. / Virology Reports RT-PCR RNA was isolated from plasma samples using QIAamp Viral RNA™ mini kit (QIAGEN®)

PT

according to the manufacturer’s instructions. Extracted dengue viral RNA was converted to

RI

cDNA using RNA amplification with a highly conserved primer pair, i.e.D1 and D2. Dengue

SC

virus sequences were amplified and typed using second-round amplification with primer D1 and four serotype-specific primers are given in Table 2 followed by according to the protocol

primers

were

used

for

ACCGGCGTCTACCCATTCATGT-3,

the

detection

MA

specific

NU

published elsewhere (Lanviotti et al .,1992). Similarly, according to the published protocol

nt10237-10258

of

CHIKV, and

(CHIKF

CHIKR

-

55-

D

GGGCGGGTAGTCCATGTTGTAGA-3, nt10544-10566) targeting a 325 base region in the

TE

cDNA encoding E1 protein of CHIKV strain 653496 (Accession # AY424803) amplify the

AC CE P

products by using thermal cycler (Master cycler, Eppendorf, Germany) (Myers et al., 1967; Khan et al., 2002). The PCR products from the above reactions were analyzed on 2% agarose gel (Fig.1 A and B). The sequences obtained were blasted against the NCBI database to ascertain the amplification of specific viral sequences. ELISA

ELISA for Dengue associated IgM was performed as per the manufacturer’s instructions (Nova Tec Immunodiagnstica GmbH, Germany). PCR Sequencing and Phylogenetic analysis of CHIKV/DENV virus

The purified PCR products of representative samples (20 DENV and 9 CHIKV) were sequenced in the forward and reverse directions using ABI PRISM 7900 sequencer (Applied Biosystems,

6

ACCEPTED MANUSCRIPT S. Dayakar et al. / Virology Reports CA, USA). The authenticity of obtained sequences was done by using the BLAST programme available of the NCBI. For sequence comparison, respective standard representative viral

PT

sequences were retrieved from GenBank and aligned with the present study sequences for the

RI

construction of phylogenetic relation using Molecular Evolutionary Genetics Analysis (MEGA

SC

6.0). (Tamura et al., 2013).Statistical analysis was performed with online epi tool 2-sample z-test

NU

to compare sample proportion.

Competing interests: None declared

D

Acknowledgements

MA

Funding: None

TE

The authors are thankful to the Indian council for Medical Research (ICMR), Govt of India

AC CE P

providing necessary infrastructure and facilities at the Viral Disease Biology program Network Grade 1 Lab at RGCB for carrying out this work. References

Arankalle, VA., Shrivastava, S., Cherian, S.,Gunjikar, RS., Walimbe, AM., Jadhav, SM., et al., 2007. Genetic divergence of Chikungunya viruses in India (1963-2006) with special reference to the 2005-2006 explosive epidemic. J Gen Virology. 88, 1967-76. Buchen, OCL., Blaine, Horzinek, MC., 2010. Virus Taxonomy. Elsevier, London, England. Debjani T, Arindam S., Bansi B., Mukhopadhyay, Shyamalendu C., 2012.A Comparative Study of Clinical Features between Monotypic and Dual Infection Cases with Chikungunya Virus and Dengue Virus in West Bengal, India. Am. J. Trop. Med. Hyg. 86(4), 720-2.

7

ACCEPTED MANUSCRIPT S. Dayakar et al. / Virology Reports John, WJ., Sevarkodiyone, SP., 2014. Spatial and temporal distribution of mosquitoes

PT

(Culicidae) in district, Tamil Nadu, South India. Inter Jour of Mosqu Rese.1 (3), 04-09. Khan, AH., Morita, K., Parquet, MD., Hasebe, F., Mathenge, EG., Lgarashi, AC.,2002.

SC

polyadenylation site. J Gen Virology. 83, 3075-84.

RI

Complete nucleotide sequence of chikungunya virus and evidence for an internal

NU

Konstantin, A., Tsetsarkin, D., Vanlandingham, L., Charles,, E. McGee., Stephen, H., 2007. A Single Mutation in Chikungunya Virus Affects Vector Specificity and Epidemic

MA

Potential. PLoS Pathogens.3(12),e201.

D

Lanviotti, RS., Calisher, CH., Gubler, DJ., Chang, GJ., Vorndam, AV., 1992. Rapid detection

TE

and typing dengue viruses from clinical samples by using Reverse Transcriptase-

AC CE P

polymerase chain reaction. J Clin Microbiol. 30, 545-51. Myers, RM., Carey, DE., 1967.

Concurrent isolation from patient of two arbo viruses,

Chickungunya and dengue type 2. Science.157, 1307-8. Tamura,

K.,

Stecher,G.,

Peterson,D.,

Filipski,A.,Kumar,S.,

2013.

MEGA6:Molecular

Evolutionary Genetic Analysis Version 6.0 Molecular Biology and Evolution. 30,27252729. Vanlandingham, DL., Hong,C., Klinger, K., Konstantin, T., Kate, LM.,, Powers, AM.,2005. Differential inactivities of o’nyong-nyong and Chickungunya virus isolates in Anopheles gambiae and Aedesaegypti mosquitoes. Am J Trop Med Hyg.72 (5), 616-21. Yergolkar, PN., Tandale, BV., Arankalle, VA., Vidya, AA., Padmakar, SS., Sudeep, AB.,2006. Chikungunya outbreaks caused by African genotype, India. Emerg Infect Dis.12, 1580-3. 8

ACCEPTED MANUSCRIPT

AC CE P

TE

D

MA

NU

SC

RI

PT

S. Dayakar et al. / Virology Reports

9

ACCEPTED MANUSCRIPT

AC CE P

TE

D

MA

NU

SC

RI

PT

S. Dayakar et al. / Virology Reports

10

ACCEPTED MANUSCRIPT

AC CE P

TE

D

MA

NU

SC

RI

PT

S. Dayakar et al. / Virology Reports

11

ACCEPTED MANUSCRIPT

AC CE P

TE

D

MA

NU

SC

RI

PT

S. Dayakar et al. / Virology Reports

12

ACCEPTED MANUSCRIPT S. Dayakar et al. / Virology Reports Table 1 Case Definitions. Symptoms

PT

Case

An acute illness characterized by sudden onset of fever with several of the following symptoms: joint pain, headache, backache, photophobia, arthralgia, rashes, etc.

Probable

Above symptoms with positive serology either when single serum sample was taken during acute onset phase or during the convalescence

NU

A confirmation was done based on the following criteria: 4-fold difference in HI antibody levels Detection of IgM antibodies against Chikungunya virus Virus isolation from plasma on cell cultures Detection of CHIKV genomic RNA by RT-PCR

AC CE P

Table 2

TE

D

1. 2. 3. 4.

MA

Confirmed

SC

RI

Suspected

Oligonucleotide primers used and the expected size of the products.

Primer DEN-F DEN-CR DEN1-R DEN2-R DEN3-R DEN4-R

sequence (5-3)

Genome position

5'-TCAATATGCTGAAACGCGCGAGAAACCG-3' 5'-TTGCACCAACAGTCAATGTCTTCAGGTTC-3' 5'-CGTCTCAGTGATCCGGGGG-3' 5'-CGCCACAAGGGCCATGAACAG-3' 5'-TAACATCATCATGAGACAGAGC-3' 5'-CTCTGTTGTCTTAAACAAGAGA-3'

Expected Size (bp)

134-161a 616-644a 568-586b 232-252b 400-421b 506-527b

511 482 119 290 392

a

The genome positions of Dl and D2 are given according to the Dengue virus type 2 published sequence (Lanviotti et al .,1992). b

The map positions of the dengue virus type-specific primers (TS1, TS2, TS3, and TS4) are given according to their respective published sequences (Lanviotti et al .,1992). 13

ACCEPTED MANUSCRIPT S. Dayakar et al. / Virology Reports Table 3

Positive for Dengue RT PCR

Dengue IgM

Positive for

by ELISA

Chikungunya

* Desired significance level

0.01

CE

16.1% (148/919) 18.8 %( 173/919) 2.3 %( 21/919)

AC

23.1%) Kerala.

32 %( 34/105)

PT ED

Andhra Pradesh 43.8 %( 46/105) 60.9 %( 64/105

MA

RT PCR

DENV vs Co-infection

CHIKV/DENV CHIKV and DENV

NU

DENV

Co infection

SC

Place infection

RI

PT

Results of Reverse transcriptase Polymerase chain reaction and ELISA tested for Dengue and Chikungunya.

14

p value (n-1024) 23 %( 24/105)

0.1% (1/919)

0 (59.9% vs 23.1%)

CHIKV vs CoCHIKV and p value (n-1024) <0.001 (34.3% vs

ACCEPTED MANUSCRIPT S. Dayakar et al. / Virology Reports Table 4

RI

PT

Detection of Dengue Serotype Specific by reverse transcriptase Polymerase chain reaction in Patient samples. a

Dengue Serotype specific RT PCR

*

NU

D-2 Vs CHIKV p value (n-189) *

<0.001 (38% vs 17.4)

Five samples we could not be genotyped, due to low copy number of DENV RNA. D-1, D-1/2, D-1/4, D-1/3, D-3/4, D-1/3/4, D-2/3/4. Desired significance level 0.01.

CE

b

64.3% (92/143) 54.3 % (25/ 46)

AC

a

35.6% (51/143) 45.6% (21/46)

PT ED

Kerala. Andhra Pradesh

Other than D-2 b

MA

D-2

SC

Place

15

Other than D2 vs CHIKV p value (n-189) * 0 (62% vs 174%)

ACCEPTED MANUSCRIPT

AC CE P

TE

D

MA

NU

SC

RI

PT

S. Dayakar et al. / Virology Reports

16