1 Immunology
MOLECULAR CHARACTERIZATION OF DENGUE AND CHIKUNGUNYA VIRUS STRAINS CIRCULATING IN NEW DELHI, INDIA [Running Title- Study of DENV & CHIKV strains from India] Nazia Afreen†a, Farah Deeba†a, Wajihul H. Khana, Shakir H. Haidera, Syed Naqui Kazima, Romana Ishrata, Irshad Hussain Naqvib, Mohammad. Y. Shareefb, Shobha Broorc±, Anwar Ahmedd , Shama Parveen*a a
Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi- 110025, India
b
Dr. M. A. Ansari Health Centre, Jamia Millia Islamia, New Delhi-110025, India
c
Department of Microbiology, All India Institute of Medical Sciences, New Delhi110029, India
d
Protein Research Chair, Department of Biochemistry, College of Science, King Saud University, Riyadh-11451,
Kingdom of Saudi Arabia
*Corresponding author address - Shama Parveen (Assistant Professor), Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi-110025, India Phone: +91-011-26984167 ext. 4492 Mobile: +91-9818286423 Email address: – [email protected] †Equal contributors Subject section –Virology, Specified field - Animal RNA virus Footnote ± Present address: Department of Microbiology, Faculty of Medicine and Health Science, Shree Guru Gobind Singh Tricentenary University, Gurgaon, Haryana-122001, India
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, Typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1348-0421.12209.
This article is protected by copyright. All rights reserved.
2 ABSTRACT Dengue and chikungunya are acute viral infections with overlapping clinical symptoms. The two diseases are transmitted by common mosquito vectors resulting in their co-circulation in a region. Molecular and serological tests specific for both dengue and chikungunya infections were carried out on 87 acute phase blood samples collected from suspected dengue/chikungunya patients in Delhi from September to December, 2011. RT-PCR and IgM ELISA were done for detection of dengue virus (DENV) and chikungunya virus (CHIKV). NS1 ELISA and IgG ELISA were also conducted for detection of DENV specific antigen and secondary DENV infection. DENV infection was detected in 49% and CHIKV infection in 29% samples by RTPCR. Co-infection by DENV and CHIKV were detected in 10% of the samples. DENV serotypes 1, 2 and 3 were detected in the study. Nine DENV-1 strains, 6 DENV-2 strains and 20 CHIKV strains were characterized by DNA sequencing and phylogenetic analysis of respective envelope protein genes. DENV-1 strains grouped in American African genotype, DENV- 2 strains in Cosmopolitan genotype and CHIKV strains in East Central South African (ECSA) genotype by phylogenetic analysis. This is one of the few studies that describe phylogeny of two dengue virus serotypes (DENV-1 and DENV-2) and chikungunya virus. The surveillance and monitoring of DENV and CHIKV strains are important for design of strategies for control of impending epidemic.
Keywords: Chikungunya, Co-infection, Dengue, India,
3 INTRODUCTION Dengue and chikungunya are arbo-viral diseases which are endemic in tropical and subtropical countries. The two diseases show many common symptoms such as fever, body ache, rash, nausea, vomiting, weakness and prostration (1, 2). Acute arthritis and rash are more prominent in chikungunya virus infection (3, 4) while dengue fever may progress to bleeding and shock due to plasma leakage (1) in some cases. The two diseases have viral aetiologies and are transmitted by common mosquito vectors i.e. Aedes aegypti and Aedes albopictus resulting in their co-circulation in a region. Dengue virus (Flaviviridae, Flavivirus), exists as four serotypes (DENV-1-4) which do not offer cross immune protection to each other (5). The four serotypes of DENV are further divided into 4-5 genotypes on the basis of their genomic sequences (6). Chikungunya viruses (CHIKV) (Togaviridae, Alphavirus) are also divided into three genotypes; Asian, West African and East/Central/South African (ECSA) (7). Thousands of cases of dengue fever are reported every year in Delhi. In the past, Delhi has witnessed dengue fever epidemics in 1967, 1970, 1982, 1988, 1996, 2003, 2006 and 2010 (8, 9, 10, 11, 12, 13, 14, 15). Many chikungunya fever cases are also reported from Delhi but the true burden of the disease is largely under-appreciated because of the similarity of its clinical symptoms with dengue fever. Therefore, molecular and serological tests specific for both dengue and chikungunya viruses were carried out in suspected samples in the present study. MATERIALS AND METHODS Sample collection The study was reviewed and approved by Institutional Ethics Committee, Jamia Millia Islamia. The study confirmed to the Declaration of Helsinki (as revised in Tokyo 2004). Acute phase blood samples were collected from suspected dengue and chikungunya patients attending Out Patient Department of Dr. M. A. Ansari Health Centre, Jamia Millia Islamia, New Delhi. The Ansari Health Centre is a small Health Centre that is located in the University campus and provides basic medical facilities to approximately 40,000 University students, employees and their dependents. Written informed consent in Hindi and English was obtained from the subjects or parent/guardian in case of minors. The age/sex, clinical symptoms and days of fever of the patients were recorded in proformas. Blood samples were transported to the Virology laboratory where sera were separated from the blood samples. Sera were stored in aliquots at -80°C until further use.
4 RNA extraction and cDNA synthesis Samples were subjected to RT-PCR for detection of DENV and CHIKV specific RNAs. RNA was extracted from serum samples using QIAamp Viral RNA Mini kit (Qiagen, Germany) according to the manufacturer’s instructions. The extracted RNA template was denatured at 65°C for 15 minutes and then reverse transcribed to cDNA in a 25 µl reaction mixture using 13.75 µL RNA, 20 ng of random primers (Promega, USA), 1mM dNTPs (Promega, USA), 8 U of rRNAsin (Promega, USA) and 20 U of Avian Myeloblastosis Virus Reverse transcriptase (Promega, USA). The reaction was allowed to proceed at 37°C for 90 minutes followed by enzyme inactivation at 65°C for 15 minutes. The cDNA thus prepared was used as template for both the DENV and CHIKV specific PCR. Diagnosis of DENV infection The DENV infection was confirmed by the presence of any of the following: (1) DENV RNA (2) DENV specific NS1 antigen (3) DENV specific antibodies (IgM/IgG). 1.
Detection of DENV RNA by RT-PCR
The RT-PCR assay employed in this study could distinguish the 4 DENV serotypes by the size of the amplicons as described by Lanciotti and colleagues (16). For first round PCR, 3 µL of the cDNA was added to 1X Taq buffer, 200µM dNTPs (Promega, USA), 0.4 µM primers (D1 and D2) and 1.5 U of Taq polymerase (Bangalore Genei, India) in 25µl reaction mixture. The PCR conditions for the external PCR were: 94°C for 2 minutes followed by 35 cycles of 94°C for 30sec, 52°C for 30sec and 72°C for 60sec and a final extension at 72°C for 10 minutes. The expected size of the PCR products was 511 base pairs (bp). External PCR was followed by nested PCR using the primer D1 and 4 serotype-specific primers, TS1, TS2, TS3 and TS4 (16). The amplified product of the external PCR was diluted in the ratio of 1:5. The 25µl nested PCR mixture was prepared by adding 1 µl of the diluted external PCR product to 1X Taq buffer, 200µM dNTPs (Promega, USA), 0.4 µM primers (D1, TS1, TS2, TS3 and TS4) and 1.5 U of Taq polymerase (Genei, India). The nested PCR was run as follows, 94°C for 1 minute followed by 25 cycles of 94°C for 30sec, 54°C for 30sec and 72°C for 60sec and a final extension at 72°C for 10 minutes. The sizes of the nested PCR products were 482 bp for DENV-1, 119 bp for DENV-2, 290 bp for DENV-3 and 392 bp for DENV-4. The PCR products were electrophoresed through 2% agarose gel, stained with ethidium bromide and examined under ultraviolet light using a gel documentation system (Wealtec, USA).
5 2.
Detection of DENV specific NS1 antigen and antibodies
NS1 antigen in the serum was detected by Dengue Early ELISA (Panbio, Australia) following manufacturers’ protocol. The test was conducted on samples of ≤4 days of illness. Serum samples with index values of >1.1 were considered positive for NS1 antigen. Index values were calculated for each sample by dividing its absorbance with cut-off value which was obtained by multiplying calibration factor specified in the kit with mean of calibrator absorbance. Presence of DENV specific IgM/IgG antibodies was detected by Dengue IgM Capture ELISA/Dengue IgG Capture ELISA (Panbio, Australia) in serum samples of ≥ 3 days of illness. In case of IgM ELISA, serum samples with index values of >1.1 were considered positive according to the kit protocol. Index values were obtained by dividing sample absorbance by cut-off value which was calculated by multiplying mean absorbance of three calibrators by calibration factor specified in the kit. The IgG ELISA kit is designed to detect elevated levels of IgG antibodies to DENV in patients with recent secondary infection. Serum samples with index values of >2.2 were considered positive for a recent secondary dengue virus infection according to the kit protocol. Index values were obtained by dividing sample absorbance by cut-off value which was calculated by multiplying mean absorbance of three calibrators by calibration factor specified in the kit. Diagnosis of CHIKV infection 1.
Detection of CHIKV infection by RT-PCR
Three μl of cDNA, published primers E1F, E1R (0.4μM) (17), 200μM of dNTPs (Promega, USA) and 1.5U Taq (Bangalore GeNei, India) in 25µl reaction, were used for the amplification of E1 protein gene. The PCR reaction was carried out with initial denaturation of 94°C for 2 minutes followed by 35 cycles of 94°C for 30sec, 52°C for 30sec and 72°C for 60sec and a final extension at 72°C for 10 minutes. The 852 bp amplicons were visualized by agarose gel electrophoresis. 2.
Detection of CHIKV specific antibodies
Serum samples with illness of ≥3days were subjected to IgM capture ELISA for CHIKV using the SD Chikungunya IgM capture ELISA kit (Standard Diagnostics, Korea) according to manufacturer’s instructions. Cut-off value was obtained by adding 0.300 in the absorbance of the negative control. DNA sequencing of envelope protein genes of DENV and CHIKV strains Serotype specific primers were used to amplify segments of E protein gene of DENV-1 and DENV-2. A 649 bp segment was amplified for DENV-1 strains using published primers (18). The PCR conditions
6 were: 94°C for 1 minute followed by 35 cycles of 94°C for 30sec, 45°C for 45sec and 72°C for 60sec and a final extension at 72°C for 10 minutes. A 574 bp segment was amplified for DENV-2 strains using published primers (19). The PCR conditions used were: 94°C for 1 minute followed by 35 cycles of 94°C for 30sec, 50°C for 60sec and 72°C for 60sec and a final extension at 72°C for 10 minutes. For CHIKV, the primers (17) used for detection were also used for sequencing of partial E1 protein gene. The amplicons were visualized on 2% agarose gel and then extracted from the gel using QIAquick Gel Extraction Kit (Qiagen, Germany). The amplicons were sequenced in both forward and reverse directions by commercial sequencing services (Ocimum, India and Xceleris Labs, India). Phylogenetic analysis of envelope protein genes of DENV and CHIKV strains The sequences were confirmed by BLAST. The forward and reverse sequences were aligned and manually edited in Genedoc (v2.7.000) software to obtain the consensus sequence. The sequences obtained in the present study and other sequences retrieved from GenBank, were aligned in ClustalW (2.1). Phylogenetic trees were constructed using Maximum Likelihood method in MEGA 6.06 software. Genetic distances were calculated using Tamura Nei model of nucleotide substitution. The robustness of the resulting tree was assessed with 1000 bootstrap replicates. The prototype strains used in the study were: Nauru strain (GenBank Accession Number U88535) for DENV-1, NGC 44 strain (GenBank Accession Number AF038403) for DENV-2 and S27 strain (Genbank Accession Number AF369024) for CHIKV. RESULTS During September to December, 2011 a total of 87 blood samples (0-10 days of illness, mean±SD: 3.47±2.77) were collected from suspected dengue and/or chikungunya patients attending Out Patient Department (OPD) of Dr. M. A. Ansari Health Centre, Jamia Millia Islamia. Severe body pain was reported by 3 patients and haemorrhagic tendency i.e. nasal bleeding, bleeding in urine was reported by 4 patients. Rest of the patients showed symptoms of fever, headache, body pain, rash, retro-orbital pain, nausea, vomiting and weakness. The mean age of patients was 25.67 years (SD ± 13.78 years) with male to female ratio as 2:1. RT-PCR RT-PCR was carried out on all 87 samples. Fifty nine (67.8%) samples were detected positive for DENV, CHIKV or both. DENV and CHIKV were detected till 10th days of illness. DENV was detected in 43
7 (49.4%) samples and CHIKV was detected in 25 (28.7%) samples. The mean age of dengue positive patients was 24.69 years (SD±14 years) while that of CHIKV positive patients was 31. 67 years (SD ±20.57 years). Twenty eight (32%) samples were found negative for both the viruses. DENV-1 was detected in 18 (42%) samples, DENV-2 in 21 (49%) samples and DENV-3 in 8 (19%) samples. DENV-4 was not detected in any of the samples. Co-infection with more than one DENV serotype was found in 4 (9.3% of the positive) samples. Concurrent infection with DENV-1 and DENV-3 was detected in two samples and co-infection by DENV-1 and DENV-2 and DENV-2 and DENV-3 were found in 1 sample each. Nine samples (10.3%) were found to be infected with both DENV and CHIKV. Mean age of concurrent DENV/CHIKV patients was 22.8 years (SD±17.97 years). Five such samples were infected with DENV-2, two samples with DENV- 3 and one sample with DENV-1. One sample showed concurrent infection by three viruses i.e. DENV-1, DENV- 2 and CHIKV. DNA sequencing and nucleotide accession numbers Nine DENV-1 and six DENV-2 partial E protein gene sequences were obtained in the study. The sequences were deposited in the GenBank database with accession numbers; KF289056-KF289064 (DENV-1 strains) and KF289065- KF289070 (DENV-2 strains). Twenty CHIKV strains were sequenced for the partial E1 protein gene. Their GenBank accession numbers are KF305686-KF305705. Two samples co-infected with DENV-1 and CHIKV viruses (DL/18/11 and DL/65/11) were sequenced for both the viruses. Phylogenetic analysis 1.
Dengue type 1 virus
The dataset used in phylogenetic analysis comprised of a total of 45 DENV-1 nucleotide sequences including 9 DENV-1 sequences obtained in the present study. The aligned region was 549bp (182 amino acid (aa)) in length spanning 342-890 bp of the E protein gene and 115-296 aa of the E protein (numbering based on the DENV-1 prototype Nauru strain, GenBank Accession Number U88535). There were 165 (30%) variable sites in the nucleotide sequence alignment while only 30 (16.5%) variable sites were detected in the deduced amino acid sequence alignment. The DENV-1 strains of the study grouped with American African genotype (Fig. 1). The DENV-1 strains sequenced in the present study showed nucleotide distance of up to 0.2% amongst themselves. These sequences showed nucleotide distance of 7% and amino acid distance of 2.2% in comparison to the prototype Nauru strain. In comparison to two
8 other Indian strains, nucleotide distance from 0.7 to 0.9% (with respect to India/10; GenBank Accession Number KC863940) and 3 to 3.2 % (with respect to India/08strain; GenBank Accession Number JF967814) were detected. Amino acid distances of 1.1% (with respect to India/10; GenBank Accession Number KC863940) and 0.6% (with respect to India/08strain; GenBank Accession Number JF967814) were detected in the study strains in comparison to two Indian strains. All the eleven study sequences showed four mutations in the aligned region i.e. Ile116Leu, Thr163Ile, Glu204Lys and Lys205Glu which are all previously reported (20). 2.
Dengue type 2 virus
The dataset used in the phylogenetic analysis comprised of a total of 51 DENV-2 sequences including 6 DENV-2 sequences obtained in the present study. The aligned region was 410bp (136aa) in length spanning 134 to 543 bp of the E protein gene and 46-181 amino acid of the E protein (numbering based on the DENV-2 prototype NGC44 strain, GenBank Accession Number AF038403). There were 160 variable sites (39%) in the nucleotide sequence alignment and 37 variable sites (27.2%) in the deduced amino acid sequence alignment. The six strains clustered within the Cosmopolitan genotype (Fig. 2). The DENV-2 strains sequenced in the present study showed 0% to 3% nucleotide distance and up to 2.2 % amino acid distance amongst themselves. These sequences showed nucleotide distances from 5.9-6.8% in comparison to the prototype NGC 44 strain, from 0.7 to 2.5% when compared to India/06 (GenBank Accession Number EU448422) strain and from 1.5 to 2.8% in comparison to India/01 (GenBank Accession Number DQ448236) strain. The study sequences showed amino acid distances of 3.7 to 4.5% in comparison to the prototype strain and 0 to 1.5% when compared to India/06 (GenBank Accession Number EU448422) strain and India/01 (GenBank Accession Number DQ448236) strain. Four mutations were found in all the studied DENV-2 strains i.e. Asp71Ala, Lys126Glu and His149Asn and Ile164Val. The mutation, Val129Ile was also found in all DENV- strains except strain DL/DENV-2/83/11. In addition, mutation Ile141Val was found in strains DL/ DENV-2/48/11, DL/DENV-2/54/11, DL/DENV2/66/11, DL/DENV-2/83/11. Another mutation Lys123Arg was found in two strains DL/DENV-2/40/11 and DL/DENV-2/72/11. All these mutations are previously reported (21). 3.
Chikungunya virus
The partial nucleotide sequences of E1 protein gene of 20 CHIKV strains were determined. The aligned region was between 616 to 1228 bp of E1 protein gene and 206 to 410 amino acids of E1 protein
9 (numbering according to prototype S27 strain, Genbank Accession Number AF369024). There were 157 variable sites (25%) found in the nucleotide sequences and 32 variable sites (15%) amongst amino acid sequences of all the CHIKV strains used in phylogenetic tree. All the study sequences clustered together within the ECSA genotype (Fig. 3). The nucleotide distance of study samples with respect to S27 strain was 3.6% and amino acid distance was 2%. With reference to the Indian strains GQ996372/08 and HM159390/09, the nucleotide divergence of 1% and 0.5% and protein divergence of up to 1% were detected respectively. The mutations identified in the analysed sequences were Lys211Glu, Met296Val, Asp284Glu and Val322Ala which have been previously reported (22). ELISA Dengue NS1 antigen was detected in 9 (16%) of the 57 samples (≤4 days of illness, mean±SD: 2±1.17) tested by NS1 ELISA. All 9 NS1 positive samples were also positive by RT-PCR. Forty eight samples were NS1 negative. Twenty NS1 negative samples were positive by RT-PCR rest 28 samples were also negative by RT-PCR. IgM ELISA and IgG ELISA for DENV were carried out on 47 samples (≥3 days of illness, mean±SD: 5.7±3.2) and 8 samples (17%) were found positive for each test. Out of IgG and IgM positive samples 4 were positive by both tests. Sample which are positive for only IgM were considered as primary infection cases whereas samples positive for IgG with or without IgM were considered as secondary infection cases. Thus primary DENV infection was detected in 4 samples whereas secondary DENV infection was detected in 8 patients. All IgG/IgM positive samples were also positive by RT-PCR except of one sample of 7 days of fever which was positive by both IgG and IgM ELISA but negative by RT-PCR. Two IgG/IgM negative samples of 3 days of fever were positive by NS1 ELISA and RT-PCR. Three samples (3 to 4 days of fever) were positive by both NS1 and IgM/IgG ELISA. There were 23 samples of ≥ 5 days of fever on which IgM and IgG ELISA were conducted. The 8 IgM positive samples included 5 samples of ≥5 days of fever and 3 samples of fever duration of 3-4 days. Chikungunya specific IgM antibodies were detected in 4 out of 40 samples (≥3 days of illness mean±SD: 5.725±2.42). All these four ELISA positive samples were negative for CHIKV infection by RT-PCR. DISCUSSION Dengue and chikungunya viruses have common mosquito vectors resulting in their co-circulation in a region. Further, these two co-circulating viruses also cause overlapping clinical symptoms in human
10 hosts. In such a scenario, testing the suspected patients for both the viruses is important for proper patient management and also for epidemiologic studies. Several reports have already described co-infection by dengue and chikungunya viruses (23, 24, 25, 4). Endemicity of dengue viruses in Delhi is well established whereas chikungunya virus is a periodically re-emerging pathogen. The present study was undertaken to conduct tests specific for both the viruses to know the prevalence of these viruses in Delhi. DENV was detected in 49% and CHIKV in 29% of the suspected patients’ samples by RT-PCR. Nine (10.3%) samples were co-infected by both the viruses. Other studies have also reported a high percentage of DENV and CHIKV co-infection cases i.e. 18.2% from Madagascar (24), 12.4% from West Bengal, India (26) and 8.7% from Delhi, India (23). Two DENV/CHIKV co-infection cases were reported from South India (27) and 5 from Delhi in 2010 (28). Detection of high number of co-infection cases in this investigation strengthens the idea that many chikungunya cases go undiagnosed in Dengue endemic regions thereby concealing the true burden of the disease. Concurrent infection with more than one DENV serotypes was also detected in 4 samples (9.3% of the positive samples). Concurrent infections were also reported in 19% of the DENV positive samples in 2006 in Delhi (29) and in 56.8% of the positive samples in Kerala in 2008 (30). Concurrent infection occurs due to co-circulation of multiple serotypes of DENV in a region. As Delhi is hyper-endemic for DENV, concurrent infections with more than one DENV serotypes are expected. Phylogenetic analysis of the sequenced DENV-1 virus strains grouped these strains in the American African genotype (Fig.1). This genotype has previously been reported from India by various authors (31, 32, 33). The study sequences clustered with strains circulating in India in 2010 (34), in Bangladesh in 2009 (Kubo et al., unpublished), in China in 2011 (Bai, unpublished) and in Singapore in 2011 (Lo et al, unpublished). The DENV-2 strains sequenced in the present study clustered within the Cosmopolitan genotype. This genotype has been previously reported from India (35, 36). In contrast to DENV-1 strains, DENV-2 strains showed more sequence diversity in gene sequence as well as protein sequence. The first group clustered with other Indian strain circulating in2006 (37) and strain from Sri Lanka (2004) (Henn et al., unpublished) (Fig. 2). The second group of sequences formed a separate cluster with strain reported from Singapore in 2010 (21). These two DENV-2 strains may have been imported from Singapore. All the sequenced CHIKV strains clustered within the ECSA genotype. These sequences showed 100% similarity to sequences reported from China in 2012 (Lu et al., unpublished) in
11 the analysed region. In addition, the study sequences were found to be 99% similar with samples reported from France (38) and Hyderabad, India (39). The A226V mutation which is considered to be responsible for the 2005-09 outbreak and because of this single mutation the virus was found to infect Aedes albopictus also along with its normal host Aedes aegypti (40) was not found in any of the study samples. NS1 antigen positivity was detected to be quite low despite conducting the test on very early acute phase samples. The low positivity indicates a low sensitivity of NS1 ELISA test used in the study. All the NS1 positive samples of the present study were positive for DENV by RT-PCR also. On the contrary, 41% of the NS1 negative samples were detected positive by RT-PCR. Similar to our data, Felix and colleagues have also reported only 38% positivity by NS1 ELISA assay on DENV RNA positive samples in Brazil in 2010 (41). Many researchers have reported a lower sensitivity of NS1 ELISA assays in secondary DENV infection patients (42, 43, 44). Contrary to our results, other studies from Delhi have shown a higher percentage of NS1 detection; 49% (45) and 66% (46%). Study subjects comprising of more primary infection patients could have been responsible for detection of higher sensitivities of NS1 ELISA in these reports (42). DENV- specific IgM and IgG antibodies were detected in 8 (17%) samples each in the present study. Similar to our results, a low sensitivity of NS1 antigen or IgM antibody test has been reported recently (47). The 4 IgG positive samples were also positive for IgM. The less seropositivity may be due to a delay in the development of adaptive immune response in the study samples. More number of secondary infection cases were detected in the study as Delhi is endemic for dengue viruses and dengue fever outbreak had occurred in 2010 (15) and 2006 (14). Four out of forty samples were found to be positive with CHIKV specific IgM capture ELISA. The less seropositivity for CHIKV in our study may be due to the presence of IgM antibodies below the detection limit (48). In conclusion, the present report describes the results of simultaneous detection of dengue and chikungunya virus infections in suspected samples from Delhi, India. Co-infection of DENV and CHIKV was detected in 10% of the study samples by RT-PCR. The study revealed circulation of three DENV serotypes (DENV- 1, 2 and 3) in Delhi in 2011. We detected lower sensitivity of NS1 ELISA in comparison to RT-PCR in first four days of illness. Phylogenetic analysis clustered DENV-1 strains of the present study in American African genotype and DENV- 2 strains within the cosmopolitan genotype. All the CHIKV strains clustered in the ECSA genotype. This is one of the few studies that describe phylogeny of two dengue virus serotypes (DENV-1 and DENV-2) and chikungunya virus. Molecular
12 characterization of circulating DENV and CHIKV strains is important for tracking the movement and evolution of these viruses so as to assist in design and implementation of control strategies. Acknowledgements This work is financially supported by research grant from University Grants Commission, Government of India. Nazia Afreen is supported by Junior Research Fellowship of the University Grants Commission, Government of India. Disclosure Authors declare no conflict of interests. References 1.
WHO (2009) Dengue Guidelines for Diagnosis, Treatment, Prevention and Control. Geneva: World Health Organization.
2.
Lahariya C., Pradhan S.K. (2006) Emergence of chikungunya virus in Indian subcontinent after 32 years: a review. J Vector Borne Dis 43: 151–160.
3.
Laoprasopwattana K., Kaewjungwad L., Jarumanokul R., Geater A. (2012) Differential diagnosis of Chikungunya, dengue viral infection and other acute febrile illnesses in children. Pediatr Infect Dis J 31: 459–463.
4.
Kularatne S.A., Gihan M.C., Weerasinghe S.C., Gunasena S. (2009) Concurrent outbreaks of Chikungunya and Dengue fever in Kandy, Sri Lanka, 2006–07: a comparative analysis of clinical and laboratory features. Post grad Med J 85: 342–346.
5.
Heinz F.X., Stiasny K. (2012) Flaviviruses and flavivirus vaccines. Vaccine 30 (29): 4301-4306.
6.
Rico-Hesse R. (2003) Microevolution and virulence of dengue viruses. Adv Virus Res 59: 315– 341.
7.
Powers A.M., Brault A.C., Tesh R.B., Weaver S.C. (2000) Re-emergence of chikungunya and o’nyong-nyong viruses: evidence for distinct geographical lineages and distant evolutionary relationships. J Gen Virol 81: 471–9.
13 8.
Balaya S., Paul S.D., D’ Lima L.V., Pavri K.M. (1969) Investigations on an outbreak of dengue in Delhi in 1967. Indian J Med Res 57: 767–774.
9.
Diesh P., Pattanayak S., Singha P., Arora D.D., Mathur P.S., Ghosh T.K., Mondal M.M., Raghavan N.G.S. (1972) An outbreak of dengue fever in Delhi-1970. J Commun Dis 4: 13–18.
10. Rao C.V.R.M., Bagchi S.K., Pinto B.D, Ilkal M.A., Bharadwaj M., Shaikh B.H., Dhanda V., Dutta M., Pavri K.M. (1985) The 1982 epidemic of dengue fever in Delhi. Indian J Med Res 82: 271–275. 11. Kabra S.K., Verma I.C., Arora N.K., Jain Y., Kalra V. (1992) Dengue haemorrhagic fever in children in Delhi. Bull World Health Organ 70: 105–108. 12. Dar L., Broor S., Sengupta S., Xess I., Seth P. (1999) The first major outbreak of dengue hemorrhagic fever in Delhi, India. Emerg Infect Dis 5(4): 589–590. 13. Singh N.P., Jhamb R., Agarwal S.K., Gaiha M., Dewan R., Daga M.K., Chakravarti A., Kumar S. (2005) The 2003 outbreak of Dengue fever in Delhi, India. Southeast Asian J Trop Med Public Health 36(5): 1174-1178. 14. Pandey A., Diddi K., Dar L., Bharaj P., Chahar H.S., Guleria R., Kabra S.K., Broor S. (2007) The evolution of dengue over a decade in Delhi, India. J Clin Virol 40: 87–88. 15. Kumari R., Kumar K., Chauhan L.S. (2011) First dengue virus detection in Aedes albopictus from Delhi, India: Its breeding ecology and role in dengue transmission. Trop Med Inter Health 16(8): 949-954. 16. Lanciotti R.S., Calisher C.H., Gubler D.J., Chang G.J., Vorndam A.V. (1992) Rapid detection and typing of Dengue Viruses from clinical samples by using Reverse Transcriptase-Polymerase Chain reaction. J Clin Microbiol 30(3): 545. 17. Santhosh S.R., Dash P.K., Parida M.M., Khan M., Tiwari M., Rao P.V.L. (2008) Comparative full genome analysis revealed E1: A226V shift in 2007 Indian Chikungunya virus isolates. Vir Res 135: 36–41.
14 18. Goncalvez A.P., Escalante A.A., Pujol F.H., Ludert J.E., Tovar D., Salas R.A., Liprandi F. (2002) Diversity and evolution of the envelope gene of dengue virus type 1. Virology 303: 110– 119. 19. Rodriguez-Roche R., Alvarez M., Gritsun T., Rosario D., Halstead S., Kouri G., Gould E.A., Guzman M.G. (2005) Dengue virus type 2 in Cuba, 1997: conservation of E gene sequence in isolates obtained at different times during the epidemic. Arch Virol 150: 415–425. 20. Warrilow D., Northill J. A., Pyke A. T. (2012) Sources of dengue viruses imported into Queensland, Australia, 2002–2010. Emerg Infect Dis 18(11): 1850. 21. Lee K.S., Lo S., Tan S.S., Chua R., Tan L.K., Xu H., Ng L.C. (2012) Dengue virus surveillance in Singapore reveals high viral diversity through multiple introductions and in situ evolution. Infect Genet Evol 12 (1): 77-85. 22. Shrinet J., Jain S., Sharma A., Singh S. S., Mathur K., Rana V., Bhatnagar R. K., Gupta B., Gaind R., Deb M., Sunil S. (2012). Genetic characterization of Chikungunya virus from New Delhi reveal emergence of a new molecular signature in Indian isolates. Virol J 9: 100. 23. Chahar H.S., Bharaj P., Dar L., Guleria R., Kabra S.K., Broor S. (2009) Co-infections with Chikungunya Virus and Dengue Virus in Delhi, India. Emerg Infect Dis 15 (7): 1077-1080. 24. Ratsitorahina M., Harisoa J., Ratovonjato J., Biacabe S., Reynes J., Zeller H., Raoelina Y., Talarmin A., Richard V., Soares J.L. (2008) Outbreak of Dengue and Chikungunya fevers, Toamasina, Madagascar, 2006. Emerg Infect Dis 14 (7): 1135-1137. 25. Leroy E.M., Nkoghe D., Ollomo B., Nze-Nkogue C., Becquart P., Grard G., Pourrut X., Charrel R., Moureau G., Ndjoyi-Mbiguino A., Lamballerie X. (2009) Concurrent Chikungunya and Dengue virus infections during simultaneous outbreaks, Gabon, 2007. Emerg Infect Dis 15(40): 591-593. 26. Taraphdar D., Sarkar A., Mukhopadhyay B.B., Chatterjee S. (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: 720–723.
15 27. Kalawat U, Sharma K.K., Reddy S.G. (2011) Prevalence of dengue and chikungunya fever and their co-infection. Indian J Path Microbiol 54(4): 844-845. 28. Singh P., Mittal V., Rizvi M.M.A., Chhabra M., Sharma P., Rawat D.S., Bhattacharya D., Chauhan L.S., Rai A. (2012) The first dominant co-circulation of both dengue and chikungunya viruses during the post-monsoon period of 2010 in Delhi, India. Epidem Infect 140: 1337–1342. 29. Bharaj P., Chahar H.S., Pandey A., Diddi K., Dar L., Guleria R., Kabra S.K., Broor S. (2008) Concurrent infections by all four dengue virus serotypes during an outbreak of dengue in 2006 in Delhi, India. Virol J 5: 1-5. 30. Anoop M., Issac A., Mathew T., Philip S., Kareem N.A., Unnikrishnan R., Sreekumar E (2010) Genetic characterization of dengue virus serotypes causing concurrent infection in an outbreak in Ernakulum, Kerala, South India. Indian J Exp Biol 48: 849–857. 31. Chakravarti A., Chauhan M.S., Kumar S., Ashraf A. (2013) Genotypic characterization of dengue virus strains circulating during 2007-2009 in New Delhi. Arch Virol 158(3): 571-581. 32. Kukreti H., Dash P.K., Parida M., Chaudhary A., Saxena P., Rautela R.S., Mittal V., Chhabra M., Bhattacharya D., Lal S., Rao P.V.L., Rai A. (2009) Phylogenetic studies reveal existence of multiple lineages of a single genotype of DENV-1 (genotype III) in India during 1956–2007. Virol J 6: 1. 33. Patil J.A., Cherian S., Walimbe A.M., Patil B.R., Sathe P.S., Shah P.S., Cecilia D. (2011) Evolutionary dynamics of the American African genotype of dengue type 1 virus in India (1962– 2005). Infect Genet Evol 11: 1443–1448. 34. Huang J. H., Su C. L., Yang C. F., Liao T. L., Hsu T. C., Chang S. F., Lin C.C., Shu, P. Y. (2012). Molecular characterization and phylogenetic analysis of dengue viruses imported into Taiwan during 2008–2010. Am J Trop Med Hygiene 87(2): 349-358. 35. Dash P.K., Sharma S., Soni M., Agarwal A., Parida M., Rao P.V. (2013) Complete genome sequencing and evolutionary analysis of Indian isolates of Dengue virus type 2. Biochem Biophys Res Commun 436(3): 478-485.
16 36. Kumar S.R.P., Patil J.A.., Cecilia D., Cherian S.S., Barde P.V., Walimbe A.M., Yadav P.D., Yergolkar P.N., Shah P.S., Padbidri V.S., Mishra A.C.,Mourya D.T. (2010) Evolution, dispersal and replacement of American genotype dengue type 2 viruses in India (1956–2005): selection pressure and molecular clock analyses. J Gen Virol 91: 707–720. 37. Shu P.Y., Su C.L., Liao T.L., Yang C.F., Chang S.F., Lin C.C., Chang M.C., Hu H.C., Huang J.H. (2009) Molecular characterization of dengue viruses imported into Taiwan during 20032007: geographic distribution and genotype shift. Am J Trop Med Hyg 80 (6): 1039-1046. 38. Grandadam M., Caro V., Plumet S., Thiberge J.M., Souares Y., Failloux A.B., Tolou H.J., Budelot M., Cosserat D., Goffart I.L., Despres P. (2011) Chikungunya Virus, Southeastern France. 2011, Emerg Infect Dis 18: 5. 39. Sumathy K., Ella K.M. (2012) Genetic diversity of chikungunya virus, India 2006–2010: Evolutionary dynamics and serotype analyses. J Med Virol 84: 462–470. 40. Santhosh S.R., Dash P.K., Parida M., Khan M., Rao P.V.L. (2009) Appearance of EI: A226V mutant Chikungunya virus in Coastal Karnataka, India during 2008 outbreak. Virol J 6:172. 41. Felix A.C., Romano C.M., Centrone C.deC., Rodrigues C.L., Villas-Boas L., AraujoE.S., de Matos A.M., Carvalho K.I., Martelli C.M., Kallas E.G., Pannauti C.S., Levi J.E. (2012) Low sensitivity of NS1 protein tests evidenced during a dengue type 2 virus outbreak in Santos, Brazil, in 2010. Clin Vaccine Immunol 19(12): 1972-1976. 42. Kumarasamy V., Chua S.K., Hassan Z., Wahab A.H.A., Chem Y.K., Mohamad M., Chua K.B. (2007) Evaluating the sensitivity of a commercial dengue NS1 antigen capture ELISA for early diagnosis of acute dengue virus infection. Singapore Med J 48 (7): 669. 43. Duong V., Ly S., Try P.L., Tuiskunen A., Ong S., Chroeung N., Lundkvist A., Leparc-Goffart I., Deubel V., Vong S., Buchy P (2011) Clinical and virological factors influencing the performance of a NS1 antigen capture assay and potential use as a marker of dengue disease severity. PLoS Negl Trop Dis 5(7): e1244.
17 44. Hang V.T., Nguyet N.M., Trung D.T., Tricou V., Yoksan S., Dung N.M., Ngoc T.V., HienT.T., Farrar J., Wills B., Simmons C.P. (2009) Diagnostic accuracy of NS1 ELISA and lateral flow rapid tests for dengue sensitivity, specificity and relationship to viraemia and antibody responses. PLoS Negl Trop Dis 3(1): e360. 45. Arya S.C., Agarwal N., Parikh S.C. (2011) Usefulness of detection of dengue NS1 antigen alongside IgM plus IgG, and concurrent platelet enumeration during an outbreak. Trans R Soc Trop Med Hyg 105: 358–359. 46. Chakravarti A., Kumar A., Malik S. (2011) Detection of dengue infection by combining the use of an NS1 antigen based assay with antibody detection. Southeast Asian J Trop Med Pub Health 42: 297-302. 47. Watthanaworawit W., Turner P., Turner C. L., Tanganuchitcharnchai A., Jarman R. G., Blacksell S. D., Nosten, F. H. (2011). A prospective evaluation of diagnostic methodologies for the acute diagnosis of dengue virus infection on the Thailand-Myanmar border. Trans Royal Soc Trop Med Hyg 105(1): 32-37. 48. Arya S.C., AgarwalN. (2011) Apropos “Outbreak of Chikungunya in the Republic of Congo and the global picture”. J Infect Dev Ctries 5(8): 609-610.
Figure legends Fig. 1. Maximum Likelihood phylogenetic tree of DENV-1 strains. The sequences obtained in the study are marked by black solid circles. The tree is based upon partial E protein gene sequences. The strains are represented by their GenBank accession number followed by country of origin and last two digits of year of isolation. The numbers on nodes represent bootstrap values generated by 1000 replications. Only bootstrap values >65 are shown. The branch lengths are proportional to the number of nucleotide changes as indicated by the scale bar (0.05 substitutions per site).
18 Fig. 2. Maximum Likelihood phylogenetic tree of DENV-2 strains. The sequences obtained in the study are marked by black solid circles. The tree is based upon partial E protein gene sequences. The tree is rooted by the sylvatic DENV-2 strains. The strains are represented by their GenBank accession number followed by country of origin and last two digits of year of isolation. The numbers on nodes represent bootstrap values generated by 1000 replications. Only bootstrap values of > 65 are shown. The branch lengths are proportional to the number of nucleotide changes as indicated by the scale bar (0.05 substitutions per site). Fig 3. Maximum likelihood Phylogenetic tree of CHIKV strains. The sequences obtained in the study are marked by solid squares. The tree is based upon partial E1 gene sequences and is rooted by the O'nyong-nyong virus strain SG650.The strains are represented by their Genbank accession numbers followed by country and year of isolation. The numbers on nodes represent bootstrap values generated by 1000 replications. The branch lengths are proportional to the number of nucleotide changes as indicated by the scale bar (0.05substitutions per site).
List of Abbreviations aa amino acid bp base pair cDNA Complementary DNA CHIKV Chikungunya Virus DENV Dengue Virus
19 dNTP Deoxy Nucleoside Triphosphate E Envelope glycoprotein ECSA East Central South East African ELISA Enzyme Linked ImmunosorbentAssay NS Non-structural protein PCR Polymerase Chain Reaction RT-PCR Reverse Transcriptase Polymerase Chain Reaction RNA Ribonucleic acid SD Standard Deviation
20
Figure 1.
21
Figure 2.
22
Figure 3.
MOLECULAR CHARACTERIZATION OF DENGUE AND CHIKUNGUNYA VIRUS STRAINS CIRCULATING IN NEW DELHI, INDIA [Running Title- Study of DENV & CHIKV strains from India] Nazia Afreen†a, Farah Deeba†a, Wajihul H. Khana, Shakir H. Haidera, Syed Naqui Kazima, Romana Ishrata, Irshad Hussain Naqvib, Mohammad. Y. Shareefb, Shobha Broorc±, Anwar Ahmedd , Shama Parveen*a a
Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi- 110025, India
b
Dr. M. A. Ansari Health Centre, Jamia Millia Islamia, New Delhi-110025, India
c
Department of Microbiology, All India Institute of Medical Sciences, New Delhi110029, India
d
Protein Research Chair, Department of Biochemistry, College of Science, King Saud University, Riyadh-11451,
Kingdom of Saudi Arabia
*Corresponding author address - Shama Parveen (Assistant Professor), Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi-110025, India Phone: +91-011-26984167 ext. 4492 Mobile: +91-9818286423 Email address: – [email protected] †Equal contributors Subject section –Virology, Specified field - Animal RNA virus Footnote ± Present address: Department of Microbiology, Faculty of Medicine and Health Science, Shree Guru Gobind Singh Tricentenary University, Gurgaon, Haryana-122001, India
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, Typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1348-0421.12209.
This article is protected by copyright. All rights reserved.
2 ABSTRACT Dengue and chikungunya are acute viral infections with overlapping clinical symptoms. The two diseases are transmitted by common mosquito vectors resulting in their co-circulation in a region. Molecular and serological tests specific for both dengue and chikungunya infections were carried out on 87 acute phase blood samples collected from suspected dengue/chikungunya patients in Delhi from September to December, 2011. RT-PCR and IgM ELISA were done for detection of dengue virus (DENV) and chikungunya virus (CHIKV). NS1 ELISA and IgG ELISA were also conducted for detection of DENV specific antigen and secondary DENV infection. DENV infection was detected in 49% and CHIKV infection in 29% samples by RTPCR. Co-infection by DENV and CHIKV were detected in 10% of the samples. DENV serotypes 1, 2 and 3 were detected in the study. Nine DENV-1 strains, 6 DENV-2 strains and 20 CHIKV strains were characterized by DNA sequencing and phylogenetic analysis of respective envelope protein genes. DENV-1 strains grouped in American African genotype, DENV- 2 strains in Cosmopolitan genotype and CHIKV strains in East Central South African (ECSA) genotype by phylogenetic analysis. This is one of the few studies that describe phylogeny of two dengue virus serotypes (DENV-1 and DENV-2) and chikungunya virus. The surveillance and monitoring of DENV and CHIKV strains are important for design of strategies for control of impending epidemic.
Keywords: Chikungunya, Co-infection, Dengue, India,
3 INTRODUCTION Dengue and chikungunya are arbo-viral diseases which are endemic in tropical and subtropical countries. The two diseases show many common symptoms such as fever, body ache, rash, nausea, vomiting, weakness and prostration (1, 2). Acute arthritis and rash are more prominent in chikungunya virus infection (3, 4) while dengue fever may progress to bleeding and shock due to plasma leakage (1) in some cases. The two diseases have viral aetiologies and are transmitted by common mosquito vectors i.e. Aedes aegypti and Aedes albopictus resulting in their co-circulation in a region. Dengue virus (Flaviviridae, Flavivirus), exists as four serotypes (DENV-1-4) which do not offer cross immune protection to each other (5). The four serotypes of DENV are further divided into 4-5 genotypes on the basis of their genomic sequences (6). Chikungunya viruses (CHIKV) (Togaviridae, Alphavirus) are also divided into three genotypes; Asian, West African and East/Central/South African (ECSA) (7). Thousands of cases of dengue fever are reported every year in Delhi. In the past, Delhi has witnessed dengue fever epidemics in 1967, 1970, 1982, 1988, 1996, 2003, 2006 and 2010 (8, 9, 10, 11, 12, 13, 14, 15). Many chikungunya fever cases are also reported from Delhi but the true burden of the disease is largely under-appreciated because of the similarity of its clinical symptoms with dengue fever. Therefore, molecular and serological tests specific for both dengue and chikungunya viruses were carried out in suspected samples in the present study. MATERIALS AND METHODS Sample collection The study was reviewed and approved by Institutional Ethics Committee, Jamia Millia Islamia. The study confirmed to the Declaration of Helsinki (as revised in Tokyo 2004). Acute phase blood samples were collected from suspected dengue and chikungunya patients attending Out Patient Department of Dr. M. A. Ansari Health Centre, Jamia Millia Islamia, New Delhi. The Ansari Health Centre is a small Health Centre that is located in the University campus and provides basic medical facilities to approximately 40,000 University students, employees and their dependents. Written informed consent in Hindi and English was obtained from the subjects or parent/guardian in case of minors. The age/sex, clinical symptoms and days of fever of the patients were recorded in proformas. Blood samples were transported to the Virology laboratory where sera were separated from the blood samples. Sera were stored in aliquots at -80°C until further use.
4 RNA extraction and cDNA synthesis Samples were subjected to RT-PCR for detection of DENV and CHIKV specific RNAs. RNA was extracted from serum samples using QIAamp Viral RNA Mini kit (Qiagen, Germany) according to the manufacturer’s instructions. The extracted RNA template was denatured at 65°C for 15 minutes and then reverse transcribed to cDNA in a 25 µl reaction mixture using 13.75 µL RNA, 20 ng of random primers (Promega, USA), 1mM dNTPs (Promega, USA), 8 U of rRNAsin (Promega, USA) and 20 U of Avian Myeloblastosis Virus Reverse transcriptase (Promega, USA). The reaction was allowed to proceed at 37°C for 90 minutes followed by enzyme inactivation at 65°C for 15 minutes. The cDNA thus prepared was used as template for both the DENV and CHIKV specific PCR. Diagnosis of DENV infection The DENV infection was confirmed by the presence of any of the following: (1) DENV RNA (2) DENV specific NS1 antigen (3) DENV specific antibodies (IgM/IgG). 1.
Detection of DENV RNA by RT-PCR
The RT-PCR assay employed in this study could distinguish the 4 DENV serotypes by the size of the amplicons as described by Lanciotti and colleagues (16). For first round PCR, 3 µL of the cDNA was added to 1X Taq buffer, 200µM dNTPs (Promega, USA), 0.4 µM primers (D1 and D2) and 1.5 U of Taq polymerase (Bangalore Genei, India) in 25µl reaction mixture. The PCR conditions for the external PCR were: 94°C for 2 minutes followed by 35 cycles of 94°C for 30sec, 52°C for 30sec and 72°C for 60sec and a final extension at 72°C for 10 minutes. The expected size of the PCR products was 511 base pairs (bp). External PCR was followed by nested PCR using the primer D1 and 4 serotype-specific primers, TS1, TS2, TS3 and TS4 (16). The amplified product of the external PCR was diluted in the ratio of 1:5. The 25µl nested PCR mixture was prepared by adding 1 µl of the diluted external PCR product to 1X Taq buffer, 200µM dNTPs (Promega, USA), 0.4 µM primers (D1, TS1, TS2, TS3 and TS4) and 1.5 U of Taq polymerase (Genei, India). The nested PCR was run as follows, 94°C for 1 minute followed by 25 cycles of 94°C for 30sec, 54°C for 30sec and 72°C for 60sec and a final extension at 72°C for 10 minutes. The sizes of the nested PCR products were 482 bp for DENV-1, 119 bp for DENV-2, 290 bp for DENV-3 and 392 bp for DENV-4. The PCR products were electrophoresed through 2% agarose gel, stained with ethidium bromide and examined under ultraviolet light using a gel documentation system (Wealtec, USA).
5 2.
Detection of DENV specific NS1 antigen and antibodies
NS1 antigen in the serum was detected by Dengue Early ELISA (Panbio, Australia) following manufacturers’ protocol. The test was conducted on samples of ≤4 days of illness. Serum samples with index values of >1.1 were considered positive for NS1 antigen. Index values were calculated for each sample by dividing its absorbance with cut-off value which was obtained by multiplying calibration factor specified in the kit with mean of calibrator absorbance. Presence of DENV specific IgM/IgG antibodies was detected by Dengue IgM Capture ELISA/Dengue IgG Capture ELISA (Panbio, Australia) in serum samples of ≥ 3 days of illness. In case of IgM ELISA, serum samples with index values of >1.1 were considered positive according to the kit protocol. Index values were obtained by dividing sample absorbance by cut-off value which was calculated by multiplying mean absorbance of three calibrators by calibration factor specified in the kit. The IgG ELISA kit is designed to detect elevated levels of IgG antibodies to DENV in patients with recent secondary infection. Serum samples with index values of >2.2 were considered positive for a recent secondary dengue virus infection according to the kit protocol. Index values were obtained by dividing sample absorbance by cut-off value which was calculated by multiplying mean absorbance of three calibrators by calibration factor specified in the kit. Diagnosis of CHIKV infection 1.
Detection of CHIKV infection by RT-PCR
Three μl of cDNA, published primers E1F, E1R (0.4μM) (17), 200μM of dNTPs (Promega, USA) and 1.5U Taq (Bangalore GeNei, India) in 25µl reaction, were used for the amplification of E1 protein gene. The PCR reaction was carried out with initial denaturation of 94°C for 2 minutes followed by 35 cycles of 94°C for 30sec, 52°C for 30sec and 72°C for 60sec and a final extension at 72°C for 10 minutes. The 852 bp amplicons were visualized by agarose gel electrophoresis. 2.
Detection of CHIKV specific antibodies
Serum samples with illness of ≥3days were subjected to IgM capture ELISA for CHIKV using the SD Chikungunya IgM capture ELISA kit (Standard Diagnostics, Korea) according to manufacturer’s instructions. Cut-off value was obtained by adding 0.300 in the absorbance of the negative control. DNA sequencing of envelope protein genes of DENV and CHIKV strains Serotype specific primers were used to amplify segments of E protein gene of DENV-1 and DENV-2. A 649 bp segment was amplified for DENV-1 strains using published primers (18). The PCR conditions
6 were: 94°C for 1 minute followed by 35 cycles of 94°C for 30sec, 45°C for 45sec and 72°C for 60sec and a final extension at 72°C for 10 minutes. A 574 bp segment was amplified for DENV-2 strains using published primers (19). The PCR conditions used were: 94°C for 1 minute followed by 35 cycles of 94°C for 30sec, 50°C for 60sec and 72°C for 60sec and a final extension at 72°C for 10 minutes. For CHIKV, the primers (17) used for detection were also used for sequencing of partial E1 protein gene. The amplicons were visualized on 2% agarose gel and then extracted from the gel using QIAquick Gel Extraction Kit (Qiagen, Germany). The amplicons were sequenced in both forward and reverse directions by commercial sequencing services (Ocimum, India and Xceleris Labs, India). Phylogenetic analysis of envelope protein genes of DENV and CHIKV strains The sequences were confirmed by BLAST. The forward and reverse sequences were aligned and manually edited in Genedoc (v2.7.000) software to obtain the consensus sequence. The sequences obtained in the present study and other sequences retrieved from GenBank, were aligned in ClustalW (2.1). Phylogenetic trees were constructed using Maximum Likelihood method in MEGA 6.06 software. Genetic distances were calculated using Tamura Nei model of nucleotide substitution. The robustness of the resulting tree was assessed with 1000 bootstrap replicates. The prototype strains used in the study were: Nauru strain (GenBank Accession Number U88535) for DENV-1, NGC 44 strain (GenBank Accession Number AF038403) for DENV-2 and S27 strain (Genbank Accession Number AF369024) for CHIKV. RESULTS During September to December, 2011 a total of 87 blood samples (0-10 days of illness, mean±SD: 3.47±2.77) were collected from suspected dengue and/or chikungunya patients attending Out Patient Department (OPD) of Dr. M. A. Ansari Health Centre, Jamia Millia Islamia. Severe body pain was reported by 3 patients and haemorrhagic tendency i.e. nasal bleeding, bleeding in urine was reported by 4 patients. Rest of the patients showed symptoms of fever, headache, body pain, rash, retro-orbital pain, nausea, vomiting and weakness. The mean age of patients was 25.67 years (SD ± 13.78 years) with male to female ratio as 2:1. RT-PCR RT-PCR was carried out on all 87 samples. Fifty nine (67.8%) samples were detected positive for DENV, CHIKV or both. DENV and CHIKV were detected till 10th days of illness. DENV was detected in 43
7 (49.4%) samples and CHIKV was detected in 25 (28.7%) samples. The mean age of dengue positive patients was 24.69 years (SD±14 years) while that of CHIKV positive patients was 31. 67 years (SD ±20.57 years). Twenty eight (32%) samples were found negative for both the viruses. DENV-1 was detected in 18 (42%) samples, DENV-2 in 21 (49%) samples and DENV-3 in 8 (19%) samples. DENV-4 was not detected in any of the samples. Co-infection with more than one DENV serotype was found in 4 (9.3% of the positive) samples. Concurrent infection with DENV-1 and DENV-3 was detected in two samples and co-infection by DENV-1 and DENV-2 and DENV-2 and DENV-3 were found in 1 sample each. Nine samples (10.3%) were found to be infected with both DENV and CHIKV. Mean age of concurrent DENV/CHIKV patients was 22.8 years (SD±17.97 years). Five such samples were infected with DENV-2, two samples with DENV- 3 and one sample with DENV-1. One sample showed concurrent infection by three viruses i.e. DENV-1, DENV- 2 and CHIKV. DNA sequencing and nucleotide accession numbers Nine DENV-1 and six DENV-2 partial E protein gene sequences were obtained in the study. The sequences were deposited in the GenBank database with accession numbers; KF289056-KF289064 (DENV-1 strains) and KF289065- KF289070 (DENV-2 strains). Twenty CHIKV strains were sequenced for the partial E1 protein gene. Their GenBank accession numbers are KF305686-KF305705. Two samples co-infected with DENV-1 and CHIKV viruses (DL/18/11 and DL/65/11) were sequenced for both the viruses. Phylogenetic analysis 1.
Dengue type 1 virus
The dataset used in phylogenetic analysis comprised of a total of 45 DENV-1 nucleotide sequences including 9 DENV-1 sequences obtained in the present study. The aligned region was 549bp (182 amino acid (aa)) in length spanning 342-890 bp of the E protein gene and 115-296 aa of the E protein (numbering based on the DENV-1 prototype Nauru strain, GenBank Accession Number U88535). There were 165 (30%) variable sites in the nucleotide sequence alignment while only 30 (16.5%) variable sites were detected in the deduced amino acid sequence alignment. The DENV-1 strains of the study grouped with American African genotype (Fig. 1). The DENV-1 strains sequenced in the present study showed nucleotide distance of up to 0.2% amongst themselves. These sequences showed nucleotide distance of 7% and amino acid distance of 2.2% in comparison to the prototype Nauru strain. In comparison to two
8 other Indian strains, nucleotide distance from 0.7 to 0.9% (with respect to India/10; GenBank Accession Number KC863940) and 3 to 3.2 % (with respect to India/08strain; GenBank Accession Number JF967814) were detected. Amino acid distances of 1.1% (with respect to India/10; GenBank Accession Number KC863940) and 0.6% (with respect to India/08strain; GenBank Accession Number JF967814) were detected in the study strains in comparison to two Indian strains. All the eleven study sequences showed four mutations in the aligned region i.e. Ile116Leu, Thr163Ile, Glu204Lys and Lys205Glu which are all previously reported (20). 2.
Dengue type 2 virus
The dataset used in the phylogenetic analysis comprised of a total of 51 DENV-2 sequences including 6 DENV-2 sequences obtained in the present study. The aligned region was 410bp (136aa) in length spanning 134 to 543 bp of the E protein gene and 46-181 amino acid of the E protein (numbering based on the DENV-2 prototype NGC44 strain, GenBank Accession Number AF038403). There were 160 variable sites (39%) in the nucleotide sequence alignment and 37 variable sites (27.2%) in the deduced amino acid sequence alignment. The six strains clustered within the Cosmopolitan genotype (Fig. 2). The DENV-2 strains sequenced in the present study showed 0% to 3% nucleotide distance and up to 2.2 % amino acid distance amongst themselves. These sequences showed nucleotide distances from 5.9-6.8% in comparison to the prototype NGC 44 strain, from 0.7 to 2.5% when compared to India/06 (GenBank Accession Number EU448422) strain and from 1.5 to 2.8% in comparison to India/01 (GenBank Accession Number DQ448236) strain. The study sequences showed amino acid distances of 3.7 to 4.5% in comparison to the prototype strain and 0 to 1.5% when compared to India/06 (GenBank Accession Number EU448422) strain and India/01 (GenBank Accession Number DQ448236) strain. Four mutations were found in all the studied DENV-2 strains i.e. Asp71Ala, Lys126Glu and His149Asn and Ile164Val. The mutation, Val129Ile was also found in all DENV- strains except strain DL/DENV-2/83/11. In addition, mutation Ile141Val was found in strains DL/ DENV-2/48/11, DL/DENV-2/54/11, DL/DENV2/66/11, DL/DENV-2/83/11. Another mutation Lys123Arg was found in two strains DL/DENV-2/40/11 and DL/DENV-2/72/11. All these mutations are previously reported (21). 3.
Chikungunya virus
The partial nucleotide sequences of E1 protein gene of 20 CHIKV strains were determined. The aligned region was between 616 to 1228 bp of E1 protein gene and 206 to 410 amino acids of E1 protein
9 (numbering according to prototype S27 strain, Genbank Accession Number AF369024). There were 157 variable sites (25%) found in the nucleotide sequences and 32 variable sites (15%) amongst amino acid sequences of all the CHIKV strains used in phylogenetic tree. All the study sequences clustered together within the ECSA genotype (Fig. 3). The nucleotide distance of study samples with respect to S27 strain was 3.6% and amino acid distance was 2%. With reference to the Indian strains GQ996372/08 and HM159390/09, the nucleotide divergence of 1% and 0.5% and protein divergence of up to 1% were detected respectively. The mutations identified in the analysed sequences were Lys211Glu, Met296Val, Asp284Glu and Val322Ala which have been previously reported (22). ELISA Dengue NS1 antigen was detected in 9 (16%) of the 57 samples (≤4 days of illness, mean±SD: 2±1.17) tested by NS1 ELISA. All 9 NS1 positive samples were also positive by RT-PCR. Forty eight samples were NS1 negative. Twenty NS1 negative samples were positive by RT-PCR rest 28 samples were also negative by RT-PCR. IgM ELISA and IgG ELISA for DENV were carried out on 47 samples (≥3 days of illness, mean±SD: 5.7±3.2) and 8 samples (17%) were found positive for each test. Out of IgG and IgM positive samples 4 were positive by both tests. Sample which are positive for only IgM were considered as primary infection cases whereas samples positive for IgG with or without IgM were considered as secondary infection cases. Thus primary DENV infection was detected in 4 samples whereas secondary DENV infection was detected in 8 patients. All IgG/IgM positive samples were also positive by RT-PCR except of one sample of 7 days of fever which was positive by both IgG and IgM ELISA but negative by RT-PCR. Two IgG/IgM negative samples of 3 days of fever were positive by NS1 ELISA and RT-PCR. Three samples (3 to 4 days of fever) were positive by both NS1 and IgM/IgG ELISA. There were 23 samples of ≥ 5 days of fever on which IgM and IgG ELISA were conducted. The 8 IgM positive samples included 5 samples of ≥5 days of fever and 3 samples of fever duration of 3-4 days. Chikungunya specific IgM antibodies were detected in 4 out of 40 samples (≥3 days of illness mean±SD: 5.725±2.42). All these four ELISA positive samples were negative for CHIKV infection by RT-PCR. DISCUSSION Dengue and chikungunya viruses have common mosquito vectors resulting in their co-circulation in a region. Further, these two co-circulating viruses also cause overlapping clinical symptoms in human
10 hosts. In such a scenario, testing the suspected patients for both the viruses is important for proper patient management and also for epidemiologic studies. Several reports have already described co-infection by dengue and chikungunya viruses (23, 24, 25, 4). Endemicity of dengue viruses in Delhi is well established whereas chikungunya virus is a periodically re-emerging pathogen. The present study was undertaken to conduct tests specific for both the viruses to know the prevalence of these viruses in Delhi. DENV was detected in 49% and CHIKV in 29% of the suspected patients’ samples by RT-PCR. Nine (10.3%) samples were co-infected by both the viruses. Other studies have also reported a high percentage of DENV and CHIKV co-infection cases i.e. 18.2% from Madagascar (24), 12.4% from West Bengal, India (26) and 8.7% from Delhi, India (23). Two DENV/CHIKV co-infection cases were reported from South India (27) and 5 from Delhi in 2010 (28). Detection of high number of co-infection cases in this investigation strengthens the idea that many chikungunya cases go undiagnosed in Dengue endemic regions thereby concealing the true burden of the disease. Concurrent infection with more than one DENV serotypes was also detected in 4 samples (9.3% of the positive samples). Concurrent infections were also reported in 19% of the DENV positive samples in 2006 in Delhi (29) and in 56.8% of the positive samples in Kerala in 2008 (30). Concurrent infection occurs due to co-circulation of multiple serotypes of DENV in a region. As Delhi is hyper-endemic for DENV, concurrent infections with more than one DENV serotypes are expected. Phylogenetic analysis of the sequenced DENV-1 virus strains grouped these strains in the American African genotype (Fig.1). This genotype has previously been reported from India by various authors (31, 32, 33). The study sequences clustered with strains circulating in India in 2010 (34), in Bangladesh in 2009 (Kubo et al., unpublished), in China in 2011 (Bai, unpublished) and in Singapore in 2011 (Lo et al, unpublished). The DENV-2 strains sequenced in the present study clustered within the Cosmopolitan genotype. This genotype has been previously reported from India (35, 36). In contrast to DENV-1 strains, DENV-2 strains showed more sequence diversity in gene sequence as well as protein sequence. The first group clustered with other Indian strain circulating in2006 (37) and strain from Sri Lanka (2004) (Henn et al., unpublished) (Fig. 2). The second group of sequences formed a separate cluster with strain reported from Singapore in 2010 (21). These two DENV-2 strains may have been imported from Singapore. All the sequenced CHIKV strains clustered within the ECSA genotype. These sequences showed 100% similarity to sequences reported from China in 2012 (Lu et al., unpublished) in
11 the analysed region. In addition, the study sequences were found to be 99% similar with samples reported from France (38) and Hyderabad, India (39). The A226V mutation which is considered to be responsible for the 2005-09 outbreak and because of this single mutation the virus was found to infect Aedes albopictus also along with its normal host Aedes aegypti (40) was not found in any of the study samples. NS1 antigen positivity was detected to be quite low despite conducting the test on very early acute phase samples. The low positivity indicates a low sensitivity of NS1 ELISA test used in the study. All the NS1 positive samples of the present study were positive for DENV by RT-PCR also. On the contrary, 41% of the NS1 negative samples were detected positive by RT-PCR. Similar to our data, Felix and colleagues have also reported only 38% positivity by NS1 ELISA assay on DENV RNA positive samples in Brazil in 2010 (41). Many researchers have reported a lower sensitivity of NS1 ELISA assays in secondary DENV infection patients (42, 43, 44). Contrary to our results, other studies from Delhi have shown a higher percentage of NS1 detection; 49% (45) and 66% (46%). Study subjects comprising of more primary infection patients could have been responsible for detection of higher sensitivities of NS1 ELISA in these reports (42). DENV- specific IgM and IgG antibodies were detected in 8 (17%) samples each in the present study. Similar to our results, a low sensitivity of NS1 antigen or IgM antibody test has been reported recently (47). The 4 IgG positive samples were also positive for IgM. The less seropositivity may be due to a delay in the development of adaptive immune response in the study samples. More number of secondary infection cases were detected in the study as Delhi is endemic for dengue viruses and dengue fever outbreak had occurred in 2010 (15) and 2006 (14). Four out of forty samples were found to be positive with CHIKV specific IgM capture ELISA. The less seropositivity for CHIKV in our study may be due to the presence of IgM antibodies below the detection limit (48). In conclusion, the present report describes the results of simultaneous detection of dengue and chikungunya virus infections in suspected samples from Delhi, India. Co-infection of DENV and CHIKV was detected in 10% of the study samples by RT-PCR. The study revealed circulation of three DENV serotypes (DENV- 1, 2 and 3) in Delhi in 2011. We detected lower sensitivity of NS1 ELISA in comparison to RT-PCR in first four days of illness. Phylogenetic analysis clustered DENV-1 strains of the present study in American African genotype and DENV- 2 strains within the cosmopolitan genotype. All the CHIKV strains clustered in the ECSA genotype. This is one of the few studies that describe phylogeny of two dengue virus serotypes (DENV-1 and DENV-2) and chikungunya virus. Molecular
12 characterization of circulating DENV and CHIKV strains is important for tracking the movement and evolution of these viruses so as to assist in design and implementation of control strategies. Acknowledgements This work is financially supported by research grant from University Grants Commission, Government of India. Nazia Afreen is supported by Junior Research Fellowship of the University Grants Commission, Government of India. Disclosure Authors declare no conflict of interests. References 1.
WHO (2009) Dengue Guidelines for Diagnosis, Treatment, Prevention and Control. Geneva: World Health Organization.
2.
Lahariya C., Pradhan S.K. (2006) Emergence of chikungunya virus in Indian subcontinent after 32 years: a review. J Vector Borne Dis 43: 151–160.
3.
Laoprasopwattana K., Kaewjungwad L., Jarumanokul R., Geater A. (2012) Differential diagnosis of Chikungunya, dengue viral infection and other acute febrile illnesses in children. Pediatr Infect Dis J 31: 459–463.
4.
Kularatne S.A., Gihan M.C., Weerasinghe S.C., Gunasena S. (2009) Concurrent outbreaks of Chikungunya and Dengue fever in Kandy, Sri Lanka, 2006–07: a comparative analysis of clinical and laboratory features. Post grad Med J 85: 342–346.
5.
Heinz F.X., Stiasny K. (2012) Flaviviruses and flavivirus vaccines. Vaccine 30 (29): 4301-4306.
6.
Rico-Hesse R. (2003) Microevolution and virulence of dengue viruses. Adv Virus Res 59: 315– 341.
7.
Powers A.M., Brault A.C., Tesh R.B., Weaver S.C. (2000) Re-emergence of chikungunya and o’nyong-nyong viruses: evidence for distinct geographical lineages and distant evolutionary relationships. J Gen Virol 81: 471–9.
13 8.
Balaya S., Paul S.D., D’ Lima L.V., Pavri K.M. (1969) Investigations on an outbreak of dengue in Delhi in 1967. Indian J Med Res 57: 767–774.
9.
Diesh P., Pattanayak S., Singha P., Arora D.D., Mathur P.S., Ghosh T.K., Mondal M.M., Raghavan N.G.S. (1972) An outbreak of dengue fever in Delhi-1970. J Commun Dis 4: 13–18.
10. Rao C.V.R.M., Bagchi S.K., Pinto B.D, Ilkal M.A., Bharadwaj M., Shaikh B.H., Dhanda V., Dutta M., Pavri K.M. (1985) The 1982 epidemic of dengue fever in Delhi. Indian J Med Res 82: 271–275. 11. Kabra S.K., Verma I.C., Arora N.K., Jain Y., Kalra V. (1992) Dengue haemorrhagic fever in children in Delhi. Bull World Health Organ 70: 105–108. 12. Dar L., Broor S., Sengupta S., Xess I., Seth P. (1999) The first major outbreak of dengue hemorrhagic fever in Delhi, India. Emerg Infect Dis 5(4): 589–590. 13. Singh N.P., Jhamb R., Agarwal S.K., Gaiha M., Dewan R., Daga M.K., Chakravarti A., Kumar S. (2005) The 2003 outbreak of Dengue fever in Delhi, India. Southeast Asian J Trop Med Public Health 36(5): 1174-1178. 14. Pandey A., Diddi K., Dar L., Bharaj P., Chahar H.S., Guleria R., Kabra S.K., Broor S. (2007) The evolution of dengue over a decade in Delhi, India. J Clin Virol 40: 87–88. 15. Kumari R., Kumar K., Chauhan L.S. (2011) First dengue virus detection in Aedes albopictus from Delhi, India: Its breeding ecology and role in dengue transmission. Trop Med Inter Health 16(8): 949-954. 16. Lanciotti R.S., Calisher C.H., Gubler D.J., Chang G.J., Vorndam A.V. (1992) Rapid detection and typing of Dengue Viruses from clinical samples by using Reverse Transcriptase-Polymerase Chain reaction. J Clin Microbiol 30(3): 545. 17. Santhosh S.R., Dash P.K., Parida M.M., Khan M., Tiwari M., Rao P.V.L. (2008) Comparative full genome analysis revealed E1: A226V shift in 2007 Indian Chikungunya virus isolates. Vir Res 135: 36–41.
14 18. Goncalvez A.P., Escalante A.A., Pujol F.H., Ludert J.E., Tovar D., Salas R.A., Liprandi F. (2002) Diversity and evolution of the envelope gene of dengue virus type 1. Virology 303: 110– 119. 19. Rodriguez-Roche R., Alvarez M., Gritsun T., Rosario D., Halstead S., Kouri G., Gould E.A., Guzman M.G. (2005) Dengue virus type 2 in Cuba, 1997: conservation of E gene sequence in isolates obtained at different times during the epidemic. Arch Virol 150: 415–425. 20. Warrilow D., Northill J. A., Pyke A. T. (2012) Sources of dengue viruses imported into Queensland, Australia, 2002–2010. Emerg Infect Dis 18(11): 1850. 21. Lee K.S., Lo S., Tan S.S., Chua R., Tan L.K., Xu H., Ng L.C. (2012) Dengue virus surveillance in Singapore reveals high viral diversity through multiple introductions and in situ evolution. Infect Genet Evol 12 (1): 77-85. 22. Shrinet J., Jain S., Sharma A., Singh S. S., Mathur K., Rana V., Bhatnagar R. K., Gupta B., Gaind R., Deb M., Sunil S. (2012). Genetic characterization of Chikungunya virus from New Delhi reveal emergence of a new molecular signature in Indian isolates. Virol J 9: 100. 23. Chahar H.S., Bharaj P., Dar L., Guleria R., Kabra S.K., Broor S. (2009) Co-infections with Chikungunya Virus and Dengue Virus in Delhi, India. Emerg Infect Dis 15 (7): 1077-1080. 24. Ratsitorahina M., Harisoa J., Ratovonjato J., Biacabe S., Reynes J., Zeller H., Raoelina Y., Talarmin A., Richard V., Soares J.L. (2008) Outbreak of Dengue and Chikungunya fevers, Toamasina, Madagascar, 2006. Emerg Infect Dis 14 (7): 1135-1137. 25. Leroy E.M., Nkoghe D., Ollomo B., Nze-Nkogue C., Becquart P., Grard G., Pourrut X., Charrel R., Moureau G., Ndjoyi-Mbiguino A., Lamballerie X. (2009) Concurrent Chikungunya and Dengue virus infections during simultaneous outbreaks, Gabon, 2007. Emerg Infect Dis 15(40): 591-593. 26. Taraphdar D., Sarkar A., Mukhopadhyay B.B., Chatterjee S. (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: 720–723.
15 27. Kalawat U, Sharma K.K., Reddy S.G. (2011) Prevalence of dengue and chikungunya fever and their co-infection. Indian J Path Microbiol 54(4): 844-845. 28. Singh P., Mittal V., Rizvi M.M.A., Chhabra M., Sharma P., Rawat D.S., Bhattacharya D., Chauhan L.S., Rai A. (2012) The first dominant co-circulation of both dengue and chikungunya viruses during the post-monsoon period of 2010 in Delhi, India. Epidem Infect 140: 1337–1342. 29. Bharaj P., Chahar H.S., Pandey A., Diddi K., Dar L., Guleria R., Kabra S.K., Broor S. (2008) Concurrent infections by all four dengue virus serotypes during an outbreak of dengue in 2006 in Delhi, India. Virol J 5: 1-5. 30. Anoop M., Issac A., Mathew T., Philip S., Kareem N.A., Unnikrishnan R., Sreekumar E (2010) Genetic characterization of dengue virus serotypes causing concurrent infection in an outbreak in Ernakulum, Kerala, South India. Indian J Exp Biol 48: 849–857. 31. Chakravarti A., Chauhan M.S., Kumar S., Ashraf A. (2013) Genotypic characterization of dengue virus strains circulating during 2007-2009 in New Delhi. Arch Virol 158(3): 571-581. 32. Kukreti H., Dash P.K., Parida M., Chaudhary A., Saxena P., Rautela R.S., Mittal V., Chhabra M., Bhattacharya D., Lal S., Rao P.V.L., Rai A. (2009) Phylogenetic studies reveal existence of multiple lineages of a single genotype of DENV-1 (genotype III) in India during 1956–2007. Virol J 6: 1. 33. Patil J.A., Cherian S., Walimbe A.M., Patil B.R., Sathe P.S., Shah P.S., Cecilia D. (2011) Evolutionary dynamics of the American African genotype of dengue type 1 virus in India (1962– 2005). Infect Genet Evol 11: 1443–1448. 34. Huang J. H., Su C. L., Yang C. F., Liao T. L., Hsu T. C., Chang S. F., Lin C.C., Shu, P. Y. (2012). Molecular characterization and phylogenetic analysis of dengue viruses imported into Taiwan during 2008–2010. Am J Trop Med Hygiene 87(2): 349-358. 35. Dash P.K., Sharma S., Soni M., Agarwal A., Parida M., Rao P.V. (2013) Complete genome sequencing and evolutionary analysis of Indian isolates of Dengue virus type 2. Biochem Biophys Res Commun 436(3): 478-485.
16 36. Kumar S.R.P., Patil J.A.., Cecilia D., Cherian S.S., Barde P.V., Walimbe A.M., Yadav P.D., Yergolkar P.N., Shah P.S., Padbidri V.S., Mishra A.C.,Mourya D.T. (2010) Evolution, dispersal and replacement of American genotype dengue type 2 viruses in India (1956–2005): selection pressure and molecular clock analyses. J Gen Virol 91: 707–720. 37. Shu P.Y., Su C.L., Liao T.L., Yang C.F., Chang S.F., Lin C.C., Chang M.C., Hu H.C., Huang J.H. (2009) Molecular characterization of dengue viruses imported into Taiwan during 20032007: geographic distribution and genotype shift. Am J Trop Med Hyg 80 (6): 1039-1046. 38. Grandadam M., Caro V., Plumet S., Thiberge J.M., Souares Y., Failloux A.B., Tolou H.J., Budelot M., Cosserat D., Goffart I.L., Despres P. (2011) Chikungunya Virus, Southeastern France. 2011, Emerg Infect Dis 18: 5. 39. Sumathy K., Ella K.M. (2012) Genetic diversity of chikungunya virus, India 2006–2010: Evolutionary dynamics and serotype analyses. J Med Virol 84: 462–470. 40. Santhosh S.R., Dash P.K., Parida M., Khan M., Rao P.V.L. (2009) Appearance of EI: A226V mutant Chikungunya virus in Coastal Karnataka, India during 2008 outbreak. Virol J 6:172. 41. Felix A.C., Romano C.M., Centrone C.deC., Rodrigues C.L., Villas-Boas L., AraujoE.S., de Matos A.M., Carvalho K.I., Martelli C.M., Kallas E.G., Pannauti C.S., Levi J.E. (2012) Low sensitivity of NS1 protein tests evidenced during a dengue type 2 virus outbreak in Santos, Brazil, in 2010. Clin Vaccine Immunol 19(12): 1972-1976. 42. Kumarasamy V., Chua S.K., Hassan Z., Wahab A.H.A., Chem Y.K., Mohamad M., Chua K.B. (2007) Evaluating the sensitivity of a commercial dengue NS1 antigen capture ELISA for early diagnosis of acute dengue virus infection. Singapore Med J 48 (7): 669. 43. Duong V., Ly S., Try P.L., Tuiskunen A., Ong S., Chroeung N., Lundkvist A., Leparc-Goffart I., Deubel V., Vong S., Buchy P (2011) Clinical and virological factors influencing the performance of a NS1 antigen capture assay and potential use as a marker of dengue disease severity. PLoS Negl Trop Dis 5(7): e1244.
17 44. Hang V.T., Nguyet N.M., Trung D.T., Tricou V., Yoksan S., Dung N.M., Ngoc T.V., HienT.T., Farrar J., Wills B., Simmons C.P. (2009) Diagnostic accuracy of NS1 ELISA and lateral flow rapid tests for dengue sensitivity, specificity and relationship to viraemia and antibody responses. PLoS Negl Trop Dis 3(1): e360. 45. Arya S.C., Agarwal N., Parikh S.C. (2011) Usefulness of detection of dengue NS1 antigen alongside IgM plus IgG, and concurrent platelet enumeration during an outbreak. Trans R Soc Trop Med Hyg 105: 358–359. 46. Chakravarti A., Kumar A., Malik S. (2011) Detection of dengue infection by combining the use of an NS1 antigen based assay with antibody detection. Southeast Asian J Trop Med Pub Health 42: 297-302. 47. Watthanaworawit W., Turner P., Turner C. L., Tanganuchitcharnchai A., Jarman R. G., Blacksell S. D., Nosten, F. H. (2011). A prospective evaluation of diagnostic methodologies for the acute diagnosis of dengue virus infection on the Thailand-Myanmar border. Trans Royal Soc Trop Med Hyg 105(1): 32-37. 48. Arya S.C., AgarwalN. (2011) Apropos “Outbreak of Chikungunya in the Republic of Congo and the global picture”. J Infect Dev Ctries 5(8): 609-610.
Figure legends Fig. 1. Maximum Likelihood phylogenetic tree of DENV-1 strains. The sequences obtained in the study are marked by black solid circles. The tree is based upon partial E protein gene sequences. The strains are represented by their GenBank accession number followed by country of origin and last two digits of year of isolation. The numbers on nodes represent bootstrap values generated by 1000 replications. Only bootstrap values >65 are shown. The branch lengths are proportional to the number of nucleotide changes as indicated by the scale bar (0.05 substitutions per site).
18 Fig. 2. Maximum Likelihood phylogenetic tree of DENV-2 strains. The sequences obtained in the study are marked by black solid circles. The tree is based upon partial E protein gene sequences. The tree is rooted by the sylvatic DENV-2 strains. The strains are represented by their GenBank accession number followed by country of origin and last two digits of year of isolation. The numbers on nodes represent bootstrap values generated by 1000 replications. Only bootstrap values of > 65 are shown. The branch lengths are proportional to the number of nucleotide changes as indicated by the scale bar (0.05 substitutions per site). Fig 3. Maximum likelihood Phylogenetic tree of CHIKV strains. The sequences obtained in the study are marked by solid squares. The tree is based upon partial E1 gene sequences and is rooted by the O'nyong-nyong virus strain SG650.The strains are represented by their Genbank accession numbers followed by country and year of isolation. The numbers on nodes represent bootstrap values generated by 1000 replications. The branch lengths are proportional to the number of nucleotide changes as indicated by the scale bar (0.05substitutions per site).
List of Abbreviations aa amino acid bp base pair cDNA Complementary DNA CHIKV Chikungunya Virus DENV Dengue Virus
19 dNTP Deoxy Nucleoside Triphosphate E Envelope glycoprotein ECSA East Central South East African ELISA Enzyme Linked ImmunosorbentAssay NS Non-structural protein PCR Polymerase Chain Reaction RT-PCR Reverse Transcriptase Polymerase Chain Reaction RNA Ribonucleic acid SD Standard Deviation
20
Figure 1.
21
Figure 2.
22
Figure 3.
