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Table of Contents
Year : 2021  |  Volume : 58  |  Issue : 4  |  Page : 386-390

Co-distribution of dengue and Chikungunya viruses in Aedes mosquitoes of Delhi, India

1 ICMR-National Institute of Malaria Research, New Delhi, India
2 South Delhi Municipal Corporation, Delhi, India

Date of Submission25-Jul-2019
Date of Acceptance10-Feb-2020
Date of Web Publication25-Mar-2022

Correspondence Address:
Ms. Rekha Saxena
Scientist- F, ICMR-NIMR, Sector-8, Dwarka, New Delhi - 110077
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0972-9062.325638

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Background & objectives: In India, vector-borne diseases, dengue and chikungunya are major public health concerns. In recent decades, dengue outbreaks have been reported in almost every part of India. In 2016, India recorded 101388 dengue cases and 210 deaths, including 4337 cases and six deaths in Delhi, whereas Chikungunya outbreaks were reported from several states in 2006, with 1.3 million cases. The Dengue virus (DENV) and Chikungunya virus (CHIKV) are both transmitted by the same Aedes mosquito species. DENV and CHIKV co-infections have been reported in 13 of 98 countries, with both viruses being transmitted locally. The reasons for the sudden upsurge in cases of these diseases are undetermined.
Methods: From March to December 2016, a study was carried out in 66 localities of Delhi in collaboration with the Municipal Corporation of Delhi. Localities were selected on the basis of confirmed dengue cases reported during the last five years and the study area was visited once a month. A door-to-door entomological survey was conducted to identify Aedes breeding in all water-filled containers in and around houses. Both immature and mature stages of Aedes mosquitoes were collected. Mosquitoes were pooled (n≤10 each for male and female) breeding site-wise and stored in Trizol at -80°C. The Chikungunya and dengue viruses were detected using a multiplex RT-PCR. Results: A total of 981 Aedes mosquitos were distributed among 146 Pools, and DENV and CHIKV were detected using Multiplex Reverse Transcriptase-PCR. Chikungunya virus was identified in 19 pools of females captured adults, whereas dengue virus was found in 8 pools of females captured adults. There was no evidence of coinfection in any of the pools.
Interpretation & conclusion: In endemic areas, continuous surveillance for both dengue and Chikungunya viruses is required to identify and characterize these viral pathogens. This information will also help implement effective strategies to combat outbreaks produced by these emerging viral pathogens.

Keywords: Dengue virus; Chikungunya virus; Coinfection; Co-distribution

How to cite this article:
Vikram K, Nagpal B N, Gupta SK, Tuli N R, Singh H, Srivastava A, Saxena R. Co-distribution of dengue and Chikungunya viruses in Aedes mosquitoes of Delhi, India. J Vector Borne Dis 2021;58:386-90

How to cite this URL:
Vikram K, Nagpal B N, Gupta SK, Tuli N R, Singh H, Srivastava A, Saxena R. Co-distribution of dengue and Chikungunya viruses in Aedes mosquitoes of Delhi, India. J Vector Borne Dis [serial online] 2021 [cited 2022 May 21];58:386-90. Available from: https://www.jvbd.org/text.asp?2021/58/4/386/325638

  Introduction Top

In India, dengue is one of the major public health problems among vector-borne diseases[1]. Dengue fever incidence has surged by 30 times in the last few decades, with more than 120 nations reporting the disease’s prev- alence[2]. In many tropical countries, the average annual incidence of the severe dengue-related disease has risen considerably in recent years, with the infection becoming endemic in cities where it was previously rare. India is a country most vulnerable to dengue outbreaks. In recent decades, dengue outbreaks have been reported in every part of the country, including Delhi. In 2016, India reported 129,166 dengue cases and 245 deaths, including 4431 cases and ten deaths in Delhi[3].

Chikungunya infection is caused by Chikungunya virus (CHIKV), which belongs to the Togaviridae family’s genus Alphavirus. More than 60 nations in Africa, Europe, Asia, the Americas, the Pacific Islands and the Indian Ocean have recorded Chikungunya cases[4]. Chikungunya was formerly not considered a life-threatening disease, but current epidemiological research reveals a 0.1% case fatality rate (Pialoux G, 2007). In 1963, India witnessed its first CHIKV outbreak, which was followed by epidemics in several regions of the country[5]. In 2006, 1.3 million chikungunya cases were recorded from various in India[6], and in 2016, India witnessed another Chikungunya outbreak with 26364 cases, including 9793 cases reported from Delhi[7].

Dengue virus (DENV) and Chikungunya virus (CHIKV) are both transmitted by the same Aedes mosquito species, Aedes aegypti; hence both viruses are likely to be spatiotemporally linked[8]. CHIKV infection has clinical symptoms that are comparable to DENV infection. The CHIKV-affected areas in Asia overlap with DENV-endemic areas[9],[10], allowing mosquitos to get infected with both viruses. DENV and CHIKV coinfections were first reported in Calcutta, India, in 1967[9]. DENV and CHIKV co-infections have been reported in 13 of the 98 countries/ territories where both viruses were transmitted locally[8].

In regions where DENV and CHKV viruses co-exist, they can be transmitted concurrently, and coinfections may be undetected owing to a lack of concurrent testing. The causes of abrupt increase in dengue and Chikungunya cases are poorly understood, although socioeconomic trends such as population expansion and urbanization are suspected. In Delhi, epidemiological data is on the co-distribution and coinfection of Chikungunya and dengue is also sparse.

  Material & Methods Top

Selection of study areas

The National Capital Territory of Delhi is 1484 square kilometres (573 sq miles). It is 51.9 km (32 miles) in length and 48.48 kilometres (30 miles) in breadth, with a population of ~17.8 million. From March to December 2016, the study was conducted in 66 Delhi localities collaborating with the Municipal Corporation of Delhi (MCD) [Figure 1]. MCD helped in the selection of all 66 localities, which were chosen based on confirmed dengue cases reported to them in the previous five years. The 66 localities includes 1) Aya Nagar, 2) Ansari Nagar, 3) Arjun Camp, 4) Badarpur, 5) Bagdola Village, 6) Basai Darapur, 7) Batla House, 8) Bharthal village (Dwarka), 9) Bijwasan Village, 10) Brijpuri (Shahdara), 11) Budh Vihar, 12) Chanakyapuri, 13) Chitra Vihar, 14) Delhi Cantt, 15) Dev Nagar, 16) East Kidwai Nagar, 17) Goyla Dairy, 18) Hari Nagar, 19) Govindpuri, 20) Indra Gandhi Nagar, 21) Jyoti Nagar (Shahdara), 22) Kakrola More, 23) Kirti Nagar, 24) Kishan Ganj, 25) Laxmibai Nagar, 26) Madhu Enclave (Najafgarh), 27) Mahavir Enclave- II, 28) Mahipalpur, 29) Mahipalpur Khurd (Airport area), 30) Manglapuri- II, 31) Maya Puri, 32) Model Town, 33) Moti Bagh, 34) Motinagar, 35) Nangal Dairy, 36) Narayana Village, 37) Nehru Vihar, 38) Netaji Nagar, 39) Palam village, 40) Palam JJ camp, 41) Pira Garhi, 42) Prem Nagar, 43) Qutub vihar, 44) Raghu Nagar, 45) Raghuvir Nagar, 46) Rajkori Village, 47) RajNagar II, 48) R. K. Puram (Sector-2), 49) Sadh Nagar, 50) Samalkha Village, 51) Sanjay Nagar (Rohini), 52) Sarai Rohilla Rly Colony, 53) Sarojini Nagar, 54) Shahabad Mohammadpur, 55) Sangam Vihar, 56) Shaheen Bagh, 57) Shakti Nagar, 58) Soniya Gandhi Camp, 59) Subhash Nagar, 60) T C Camp Najafgarh, 61) Tagore Garden, 62) Trilokpuri, 63) Om Vihar (Uttam Nagar), 64) Vasant Kunj, 65) Vijay Vihar (Rohini), 66) West Sagarpur. During the study period, about 15000 households were surveyed, with an average of 227 households inspected in each locality. The study areas were visited once a month.
Figure 1: Map of Delhi, India showing 66 localities included in the study

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Entomological surveys

Aedes breeding in all types of water-filled containers present within houses and their premises was carried out during a door-to-door entomological survey conducted in 66 localities. Aedes mosquitoes were obtained in both immature and mature stages. The larval collections were made using flash-light, dipping, and pipetting[11]. The presence of immature stages of Aedes mosquitoes was screened in all types of breeding sites like curing tanks, overhead tanks, various types of plastic containers (drums/ tanks/ tubs), coolers, solid waste (disposal glass, thermocol pots, washbasin, tire dumps, booster pumps, cement pots, iron pipes, plastic sheets, plastic trays, iron pots, plastic chairs, trollies, disposal pots, fireboxes, dustbins, discarded almirahs, plastic buckets), and others (flower pots, junk materials, broken glassware, bottles). For species identification, all larvae and pupae were reared to the adult stage, and further, Aedes aegypti mosquitoes were separated based on their sex and collecting sites. Mosquitoes were pooled by breeding location (n≤10 each for male and female) and preserved in Trizol at -80°C until further use.

Detection of dengue and Chikungunya viruses with Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)

We used the QIAamp Viral RNA™ mini kit (QIA-GEN®) to isolate RNA from mosquito pools according to the manufacturer’s instructions. In 25 μl total reaction volume, one-step multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) was performed using a highly conserved primer pair for dengue, i.e., D1 (5’-TCAATATGCTGAAACGCGCGAGAAACCG-3’) and D2 (5’-TTGCACCAACAGTCAATGTCTTCAG-GTTC-3’) to anneal to any of the four dengue virus serotypes that amplifies a 511-bp product in RT-PCR [12].

Similarly, specific primers were used for the detection of CHIKV (CHIKF - 5′-ACCGGCGTCTACCCATTCAT- GT-3′, nt10237-10258 and CHIKR - 5′-GGGCGGG- TAGTCCATGTTGTAGA-3′, nt10544-10566) targeting a 325 base region in the cDNA encoding E1 protein of CHIKV strain 653496 (Accession # AY424803) amplify the products with the use of thermal cycler (Master cycler, Eppendorf, Germany)[13].

Thermal cycling of RT-PCR was 48°C for 30 min and 94°C for 2 min, followed by 35 cycles of 94°C for 1 min, 54°C for 1 min and 72°C for 1 min and a final extension cycle at 72°C for 10 min. RT-PCR products were detected by electrophoresis in 2% agarose gel.

  Results Top

A total 981 Aedes mosquitoes (614 field-collected and 367 emerged) were distributed in 146 Pools (102 pools of captured adults and 44 pools of emerged adults). Out of 102 pools of captured adults, 63 pools of females and 39 pools of males were distributed, whereas out of 44 pools of emerged adults, 21 pools of females and 23 pools of males were distributed. DENV and CHIKV were tested in all 146 pools of Aedes mosquitoes using Multiplex Reverse Transcriptase-PCR.

[Figure 2] shows agarose gel electrophoresis of Multiplex Reverse Transcriptase-PCR of CHIKV and DENV. The DNA product size of 325-bp was obtained for each positive pool after amplification with chikungunya primers, and 511 bp size of DNA product was obtained for the positive pool after amplification with dengue primers.
Figure 2: Multiplex RT-PCR based detection of DENV and CHKV in the mosquito samples. M-100 bp Marker, Lane 1,2,9,10-CHK Positive (325 bp), Lane 4- DEN Positive (511 bp), Lane 3,5-8- Negative samples

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Chikungunya virus was detected in 19 pools of female adults from Aya Nagar, Bagdola Village, Brijpuri, Budh Vihar, East Kidwai Nagar, Hari Nagar, Laxmibai Nagar, Manglapuri-II, Nangal Dairy, Palam Village, Prem Nagar, Raghubir Nagar, Raj Nagar-II, R. K. Puram (Sector-2), Sarai Rohilla Rly Colony, Soniya Gandhi Camp, Om Vihar (Uttam Nagar), and Vijay Vihar, while dengue virus was discovered in 8 pools of female adults from Aya Nagar, Budh Vihar, Goyla Dairy, Jyoti Nagar, Mahavir Enclave-II, Mahipalpur, R. K. Puram (Sector-2) and Sangam Vihar. DENV and CHIKV were reported to be negative in any pool of males or emerging adults (females and males). There was no coinfection in any pool; however, there was co-distribution in three locations: Aya Nagar, Budh Vihar, and R. K. Puram.

  Discussion Top

Dengue and Chikungunya viruses are transmitted through a common vector i.e., Aedes aegypti and Aedes albopictus which mainly contributes towards co-circulation and coinfection of these viruses in the mosquito. Following a mosquito bite, these viruses are transferred to humans as coinfections. The findings of this study suggest that DENV and CHIKV are co-distributed in Delhi; however, no coinfection was found in any pool. This study showed that both DENV and CHIKV could not multiply in the same mosquito concurrently, consistent with the previous study. The absence of coinfection with both viruses indicates a viral interference problem[14]. The co-circulation of two or more mosquito-borne flaviviruses and alphaviruses in the same geographic area increases the opportunity for viral interactions, resulting in viral interference[15]. The primary virus infection possibly induces a host-immune response that suppresses a secondary viral infection[16]. The effect of coinfection of arboviruses on virus replication is still unclear. The present study results are inconsistent with previous studies that reported the presence of both viruses in Ae. albopictus[17],[18] and Ae. aegypti[19] suggesting coinfection from DENV and CHIKV.

Similarly, coinfections in Ae. aegypti with distinct arboviruses i.e., DENV, CHIKV and ZIKV were reported in other investigations[16],[20]. The detection of dengue and Chikungunya virus coinfection in Aedes mosquito suggests the possibility that people might be infected with both viruses by a single mosquito bite or two consecutive infectious blood meals from two distinct viremic hosts[17].

More mosquito pools tested positive for the Chikungunya virus compared to the dengue virus, indicating that the Chikungunya virus multiplies rapidly in Aedes mosquitos. The variation in incubation periods of these viruses in mosquito vectors supports this[21]. Furthermore, the present study did not provide any evidence for vertical transmission of DENV and CHIKV, which has been observed in the natural population of Aedes aegypti in Mexico[22]. In Martinique, the dengue-chikungunya outbreak was reported in 2013–2015, during which vertical transmission of DENV was observed in Aedes aegypti mosquito[19]. The mechanism of coinfection in mosquito vectors by two distinct arboviruses has to be researched further to discover whether this is a common phenomenon or an exception due to predominantly well-altered partners.

  Conclusion Top

Repeated dengue outbreaks, current CHIKV activity, and CHIKV/DENV coinfections in Delhi show a shift in the epidemiology of these viruses in the region. Due to the overlap of many signs and symptoms, it can be difficult to distinguish between the two infections clinically, making diagnosis and treatment challenging. As a result, in the endemic areas of Delhi, continuous and rigorous virological surveillance for DENV and CHIKV is required not only to identify and characterize these viruses but also to implement effective control strategies to prevent future infection outbreaks caused by these emerging viruses.

Conflict of Interest: None

  Acknowledgment Top

Authors are thankful to ICMR-NIMR for funding support. Special thanks to MCD for helping us in selection of study sites in Delhi. We are thankful to the entire field staff of NIMR for assistance in field work. We thank Mr. Mritunjay Prasad Singh for data entry and data management. This paper bears the NIMR publication screening committee approval no. 61/ 2019.

  References Top

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  [Figure 1], [Figure 2]


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