Co-infections with Chikungunya Virus and Dengue Virus in Delhi, India

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DOI: 10.3201/eid1507.080638 Suggested citation for this article : Chahar HS, Bharaj P, Dar L, Guleria R, Kabra SK, Broor S. Co-infections with chikungunya virus and dengue virus in Delhi, India. Emerg Infect Dis.

2009 Jul; [Epub ahead of print] Co-infections with Chikungunya Virus and Dengue Virus in Delhi, India Harendra S. Chahar, Preeti Bharaj, Lalit Dar, Randeep Guleria, Sushil K. Kabra, and Shobha Broor Author affiliation: All India Institute of Medical Sciences, New Delhi, India Aedes aegypti mosquitoes are common vectors for dengue virus and chikungunya virus.

In areas where both viruses cocirculate, they can be transmitted together. During a dengue outbreak in Delhi in 2006, 17 of 69 serum samples were positive for chikungunya virus by reverse transcription 3PCR; 6 samples were positive for both viruses. Chikungunya virus (CHIKV) was isolated in Tanganyika (now Tanzania) in 1953 ( 1 ).

In Asia, this virus is transmitted almost exclusively by Aedes aegeypti mosquitoes. India had its first CHIKV outbreak in 1963; it was followed by epidemics in other parts of the country ( 2 ). Recently, massive outbreaks of CHIKV have been reported from many islands in the Indian Ocean ( 3 ).

Chikungunya outbreaks in India were reported in 2005, and 1.4 million ... more. less.

chikungunya cases were reported from different states ( 3 ). Estimated annual incidence of disease caused by dengue virus (DENV) is 50 3100 million cases of dengue fever and 250,000 cases of dengue hemorrhagic fever; mortality rate is 25,000 per year in tropical and subtropical countries. Like CHIKV, DENV is also transmitted by Ae . aegypti and is endemic to urban and semiurban areas of India ( 4 ). In Asia, the CHIKV-affected areas overlap with DENV-endemic areas ( 5 , 6 ) and provide opportunities for mosquitoes to become infected with both viruses. Co-infection with 2 dengue viruses (DENV-1 and DENV-4) was reported in Puerto Rico in 1982 ( 7 ). Since then, many cases Page 1 of 9 of concurrent infections with multiple DENV serotypes have been reported in many countries. Since 2005, co-infections with >2 DENV serotypes have been reported in Delhi, India ( 8 ). Co- infections with DENV and CHIKV were reported in Calcutta, India, in 1967 ( 5 ). Subsequent serologic investigations in southern India indicated that the 2 viruses can coexist in the same host ( 9 ). We report detection by reverse transcription 3PCR of co-infections with CHIKV and DENV in clinical samples obtained during the 2006 dengue outbreak in Delhi, India. The Study Acute-phase blood samples were collected from 69 patients with clinically suspected cases of DENV/CHIKV co-infection. Viral RNA was extracted from serum samples by using the MagNA Pure Compact Nucleic Acid Isolation System (Roche Diagnostics, Basel, Switzerland). Published primers and cycling conditions were used for the amplification of DENV ( 10 ) and CHIKV ( 11 ). cDNA was synthesized by using avian myeloblastosis virus reverse transcriptase (Promega Corp., Madison, WI, USA) and downstream consensus primer (D2) for DENV and random hexamers for CHIKV. DENV typing was performed by using second-round amplification with type-specific primers ( 10 ). The amplified products were visualized by electrophoresis on 2% agarose gels. Because samples were received during a dengue outbreak, ethical clearance was not required. Partial nucleotide sequences of the envelope 1 (E1) gene (294 bp) of CHIKV were determined by using an automated 310 DNA sequencer (Applied Biosystems Inc., Foster City, CA, USA). Sequences were aligned, analyzed, subjected to homology search by BLAST analysis ( www.ncbi.nlm.nih.gov/Education/BLASTinfo/information3.html ), and submitted to GenBank (accession nos. EU727159 363 and EF539265). Phylogenetic analysis of CHIKV sequences (Table) was conducted by using ClustalW ( www.ebi.ac.uk/Tools/clustalw2/index.html ) and MEGA version 3.1 software ( 12 ), Kimura 2-parameter distances, and neighbor-joining algorithms. Of 69 samples tested, DENV RNA was detected in 48 and CHIKV RNA in 17. Of the 17 CHIKV-positive samples, 6 were co-infected with DENVs. Three of the 6 samples from patients co-infected with CHIKV/DENV contained DENV-3; 1 contained DENV-4, and 2 contained 2 Page 2 of 9 DENV serotypes (1 contained DENV-3 and DENV-4 and 1 contained DENV-3 and DENV-1) (Figure 1). Of the 17 CHIKV-positive patients, 10 were male and 6 were female. Information regarding age, sex, and clinical features was not available for 1 patient. Thirteen samples were from adults (>12 years of age) and 3 were from children (< 12 years of age). Retrospective analysis of medical records identified clinical information for 6 patients co- infected with DENV and CHIKV. All 6 patients had fever, headache, joint pain, and low thrombocyte counts (<100,000/mm 3 ). The patients with only CHIKV infection had fever, headache, and joint pain. Of the 6 patients with co-infections, 2 had dengue hemorrhagic fever with central nervous system (CNS) involvement. CNS involvement and hemorrhagic manifestations may be caused by DENVs because these manifestations are common in patients infected with DENV; CNS involvement has been documented in persons with DENV infections ( 13 ). In 2 patients with CNS involvement, 1 was infected with DENV-3 and 1 was infected with DENV-4. Of the 6 patients with co-infections, 5 fully recovered and 1 died. Phylogenetic analysis of partial E1 gene sequences demonstrated that all CHIKV strains from Delhi grouped with isolates obtained during 2006 from southern India and islands in the Indian Ocean and belonged to the Central East African genotype (Figure 2). This finding indicates that during 2006 similar strains were circulating throughout India. Isolates obtained in India during 1963 31973 clustered with isolates from Thailand (Thai 62 378) and formed a separate cluster in the Asian genotype. Conclusions For many years, it appeared that CHIKV had disappeared from India, but late in 2005 the virus reemerged on Reunion Island and in India ( 3 ). Confirmed cases of CHIKV infection have been reported from Delhi, Haryana, Uttar Pradesh, and Rajasthan provinces in northern India, although these states did not have large-scale epidemics ( 14 ). DENV infections are endemic to northern India; in recent years, increasing trends of cocirculation of multiple DENV serotypes in Delhi suggest that DENVs are becoming hyperendemic to this region ( 8 ). During 2006, DENV and CHIKV were detected in Delhi ( 14 ). Because the clinical features of DENV and CHIKV are similar, CHIKV infections may go Page 3 of 9 undiagnosed in DENV-endemic areas. In India, Ae . aegypti mosquitoes are primary vectors for DENV and CHIKV, and opportunities for co-infections in humans are increased by the feeding behavior of the mosquito ( 15 ), low socioeconomic conditions, and high population density. We report co-infections with DENV and CHIKV in India after a long absence of the viruses in this region. It is difficult to comment on increased severity of illness in patients with DENV/CHIKV co-infections because the number of patients tested was small. Additional clinical information is needed to determine the influence of co-infections on clinical expression of dengue and chikungunya fever. Our study indicates that co-infections with CHIKV and DENV occur in areas where these 2 viruses cocirculate. Concurrent infections may result in illness with overlapping signs and symptoms, making diagnosis and treatment difficult for physicians. Repeated outbreaks of dengue, recent activity of CHIKV, and CHIKV/DENV co-infections in the Delhi area suggest that the epidemiology of these viruses is changing in this region and that these viruses are becoming endemic to this region. Thus, in clinically suspected cases of dengue or chikungunya fever, it is advisable to test for both viruses in areas where they cocirculate. Acknowledgments We thank Ashok Saini, Salek Chand, and Devender Kumar for excellent technical support; and Chandra P. Prasad and Sailesh Bajpai for critically reading the manuscript and making valuable suggestions. This study was supported by All India Institute of Medical Sciences and the government of India. H.S.C. was supported by a fellowship from the Indian Council of Medical Research. Mr Chahar is a doctoral student at the All India Institute of Medical Sciences in New Delhi, India. His research interest is the development of silent RNA 3based approaches for inhibition of multiplication of respiratory syncytial virus and human metapneumovirus. References 1. Ross RW. The Newala epidemic III; the virus: isolation, pathogenic properties and relationship to the epidemic. J Hyg (Lond). 1956;54:177 391. PubMed 2. Shah KV, Gibbs CJ Jr, Banerjee G. Virological investigation of the epidemic of haemorrhagic fever in Calcutta: isolation of three strains of chikungunya virus. Indian J Med Res. 1964;52:676 383. PubMed Page 4 of 9 3. Ravi V. Re-emergence of chikungunya virus in India. Indian J Med Microbiol. 2006;24:83 34. PubMed 4. Gubler DJ. Dengue. In: Monath TP, editor. The arboviruses: epidemiology and ecology. Vol. II. Boca Raton (FL): CRC Press; 1988. p. 223 3260. 5. Myers RM, Carey DE. Concurrent isolation from patient of two arboviruses, chikungunya and dengue type 2. Science. 1967;157:1307 38. PubMed DOI: 10.1126/science.157.3794.1307 6. Mackenzie JS, Chua KB, Daniels PW, Eaton BT, Field HE, Hall RA, et al. Emerging viral diseases of Southeast Asia and the Western Pacific. Emerg Infect Dis. 2001;7:497 3504. PubMed 7. Gubler DJ, Kuno G, Sather GE, Waterman SH. A case of natural concurrent human infection with two dengue viruses. Am J Trop Med Hyg. 1985;34:170 33. PubMed 8. Bharaj P, Chahar HS, Pandey A, Diddi K, Dar L, Guleria R, et al. Concurrent infections by all four dengue virus serotypes during an outbreak of dengue in 2006 in Delhi, India. Virol J. 2008;5:1. PubMed DOI: 10.1186/1743-422X-5-1 9. Yergolkar PN, Tandale BV, Arankalle VA, Sathe PS, Sudeep AB, Gandhe SS, et al. Chikungunya outbreaks caused by African genotype, India. Emerg Infect Dis. 2006;12:1580 33. PubMed 10. Lanciotti RS, Calisher CH, Gubler DJ, Chang GJ, Vorndam AV. Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase 3polymerase chain reaction. J Clin Microbiol. 1992;30:545 351. PubMed 11. Hasebe F, Parquet MC, Pandey BD, Mathenge EG, Morita K, Balasubramaniam V, et al. Combined detection and genotyping of chikungunya virus by a specific reverse transcription 3polymerase chain reaction. J Med Virol. 2002;67:370 34. PubMed DOI: 10.1002/jmv.10085 12. Kumar S, Tamura K, Nei M. MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform. 2004;5:150 363. PubMed DOI: 10.1093/bib/5.2.150 13. Lum LC, Lam SK, Choy YS, George R, Harun F. Dengue encephalitis: a true entity? Am J Trop Med Hyg. 1996;54:256 39. PubMed 14. Directorate General of Health Services Ministry of Health and Family Welfare. National Vector Borne Disease Control Program. State-wise status of chikungunya fever in India, 2006. [cited 2008 May 10]. Available from http://www.nvbdcp.gov.in/Doc/CHK.pdf Page 5 of 9 15. Scott TW, Naksathit A, Day JF, Kittayapong P, Edman JD. A fitness advantage for Aedes aegypti and the viruses it transmits when females feed only on human blood. Am J Trop Med Hyg. 1997;57:235 39. PubMed Address for correspondence: Shobha Broor, Virology Division, Department of Microbiology, Rm 2105, All India Institute of Medical Sciences, New Delhi 110029, India; email: shobha.broor@gmail.com Page 6 of 9 Table. Chikungunya virus sequences, including strains from Delhi and southern India, used for phylogenetic analysis* Sequence no. Laboratory ID or isolate name Year State/country GenBank accession no. 1 GOA 018 2006 Goa/India EF187902 2 HYD 349 2006 Hyderabad/India EF187893 3 GWL 008 2006 Madhya Pradesh/India EF187904 4 HYR023 2006 Karnataka/India EF187899 5 CHTR 54 2006 Andhra Pradesh/India EF187897 6 IND06 AP5 2006 Andhra Pradesh/India DQ520744 7 IndKL 01 2006 Kerala/India EU119154 8 IND06 MH1 2006 Maharashtra/India DQ520734 9 IND06 AP6 2006 Andhra Pradesh/India DQ520745 10 IND06 MS2 2006 Andhra Pradesh/India DQ520740 11 IND06 MS1 2006 Andhra Pradesh/India DQ520741 12 IND06 KA3 2006 Karnataka/India DQ520738 13 PON1 2006 Pondicherry/India EF113095 14 IND05 KA1 2005 Karnataka/India DQ520737 15 REUNION 2006 Réunion Island DQ443544 16 IND06 AP3 2006 Andhra Pradesh/India EF027134 17 IND06 MH2 2006 Maharashtra/India EF027136 18 IND06 MH3 2006 Maharashtra/India DQ520736 19 ROSS 1953 Tanzania AF490259 20 TAN53 1953 Tanzania AF192905 21 IND00 MH4 2000 Maharashtra/India EF027139 22 CONGO02 2000 Congo AY549581 23 CONGO03 2000 Congo AY549579 24 CONGO01 2000 Congo AY549583 25 S27AFRICA 1953 Tanzania NC004162 26 MALAYA98A 1998 Malaysia AF394210 27 MALAYA98B 1998 Malaysia AF394211 28 THAI95 1995 Thailand AF192897 29 THAI96 1996 Thailand AF192900 30 THAI88 1988 Thailand AF192896 31 PHILLIP85 1985 The Philippines AF192895 32 INDON85 1985 Indonesia AF192894 33 THAI75 1975 Thailand AF192898 34 THAI78 1978 Thailand AF192899 35 THAI62 1962 Thailand AF192908 36 IND71CH1 1971 Tamil Nadu/India DQ520751 37 IND63WB1 1963 West Bengal/India EF027140 38 IND64CH2 1964 Tamil Nadu/India DQ520748 39 SENEG66 1966 Senegal AF192891 40 NIGER64 1963 Nigeria AF192893 41 SENEG83A 1983 Senegal AY726732 42 SENEG83B 1983 Senegal AF192892 43 O 9NYONG-NYONG 1996 Uganda AF079456 44 DEL/1467/06 2006 Delhi/India EF539265 45 DEL/758/06 2006 Delhi/India EU727160 46 DEL/868/06 2006 Delhi/India EU727163 47 DEL/968/06 2006 Delhi/India EU727161 48 DEL/1307/06 2006 Delhi/India EU727162 49 DEL/1795/06 2006 Delhi/India EU727159 *ID, identification no. Page 7 of 9 Figure 1. Agarose gel electrophoresis showing chikungunya virus (A) and dengue virus (B) PCR products. A) Lane 1, 294-bp product specific for chikungunya virus; lane 2, 100-bp DNA marker. B) Lane 1, 100-bp DNA marker; lane 2, 119-bp product specific for dengue 2 virus; lane 3, 290-bp product for dengue 3 virus and 392-bp product for dengue 4 virus. Page 8 of 9 Figure 2. Phylogenetic analysis of partial envelop 1 (E1) gene sequences (294 bp) of chikungunya virus strains from the 2006 dengue outbreak in Delhi, India. Neighbor-joining tree was constructed by using E1 gene sequences from various chikungunya virus sequences. O 9nyong-nyong virus (AF079456) was used as an outgroup. Percentage bootstrap support is indicated by the values at each node. Delhi strains are indicated by a diamond. Scale bar indicates 0.02 nucleotide substitutions per site. Page 9 of 9

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