Initially the surveillance program was carried out in proposed high-risk areas in Kerala state, Tamil Nadu and Puducherry (2016-2017) by ICMR-Vector Control Research Centre. Subsequently, it was extended to all the high-risk zones of the Country covering 49 Districts distributed to 14 states / Union Territories of the Country (Fig. 1 & Table 1).
Study Area
All the institutions involved were offered with a weeklong training program on the uniform Standard Operating protocol (SOP) to be adopted for the project studies. Regions for sampling of Aedes specimens invariably included high-risk zones as international entry sites such as International Airports, Seaports and national ones like Airports, metropolitan cities, urban corporations, major rail stations, major bus terminals etc. Aedes mosquitoes were sampled on weekly intervals. The collections were planned in co-operation with local State health Department machinery. However, the modalities varied from state to state. The proposed methodology was as follows:
Adult Aedes mosquito collections
Resting mosquitoes indoor were collected using vacuum aspiration in early morning hours (8.00-10.00 hrs). A battery operated prokopack or handheld mechanical aspirators were used to collect mosquitoes resting in and around houses. The specimens from aspirators were transferred to Barraud cages or to test tubes (one specimen in a test tube).
Mosquitoes attempting to bite was sampled by lure baited Bio Gents (BG) sentinel traps set for about 24 hours in morning hours set indoors and outdoors. The specimens collected in BG sentinel traps were collected next day. Gravid traps collected mosquitoes seeking habitats for egg laying. Gravid traps were set in the morning hours and specimens trapped were recovered daily in the early morning. Sweep nets were used to collect outdoor resting mosquitoes. All collected live Aedes mosquitoes were brought to the laboratory, stunned (either keeping in a freezer for 20 minutes or by hand tapping) and identified morphologically using standard taxonomic keys. They were sorted by sex, species, collection method, and collection period and pooled to a maximum no. of 20 identical category specimens and were placed into 2 ml eppendorff tubes with 50µl TRI reagent (MRC, USA) and were stored at 4o Celsius. These tubes were transported to the ICMR institutions once a week, where it was further processed for arbo-viral infection status.
Immature Aedes collections
All indoor and outdoor water-containing receptacles at the selected households were inspected for Aedes mosquito larvae and pupae. Live larvae observed in receptacles were collected and reared to adults towards identification. All the pupae found were collected were also reared to adulthood. On emergence, the specimens were sorted according to species and gender and were pooled (maximum 20 specimens) to Eppendorf tubes with 50-µl TRI reagent. These were also stored at 4o C and were transported to ICMR institution for further processing.
RNA Extraction from mosquito pools
The entire procedure was carried out in BSL-Class 2 facilities. Pooled specimens were homogenized in Eppendorf tubes thoroughly using Kontes pellet pestle motor until no visible parts remained. It was made up to 200µl with TRI reagent (1-2 specimens). (100µl of additional TRI reagent for each additional specimen in the pool and for a pool of 10 specimens required quantity of TRI reagent was 1ml). The homogenate was incubated for 5 min at RT, added 200 µl of chloroform (for 1 ml homogenate) and vortexed for 15 sec. Further, the reaction mixture was incubated at room temperature for 15 min. and centrifuged at 12,000 g for 15 min at 4°C.
The aqueous upper layer was pipetted out and was transferred to a fresh tube. 500 µl of isopropanol was added and stored at RT for 6-7 min. Centrifuged at 12,000g for 8 min (4°C). Discarded the supernatant by tilting the tube gently. Washed pellet twice with ice-cold 75% and RT 100% ethanol. Air dried the pellet at RT and dissolved in 30 ul de-ionized water.
RT- PCR for detection of DENV/ZIKV infections
One-step Roche transcriptor kit was used for the multiplex RT-PCR reactions. Partial envelope gene of ZIKV and CprM gene of DENV were amplified by performing a multiplex PCR [21,22].
The primers used are as follows:
ZIKA Virus infection:
ZIKVENVF (5’-GCTGGDGCRGACACHGGRACT-3’)
ZIKVENVR (5’-RTCYACYGCCATYTGGRCTG-3’)
Dengue Virus infection
Dl 5'-TCAATATGCTGAAACGCGCGAGAAACCG-3'
D2 5'-TTGCACCAACAGTCAATGTCTTCAGGTTC-3'
25 µl PCR reactions were setup, which consisted of 5 µl Buffer, 1 µl each of all the 4 DNA primers cited above, 0.5 µl enzyme mix, template 2 µl and 13.5 µl water supplied with the kit. DENV and ZIKV positive control and a Negative Control were included in all the reactions.
The reaction conditions was an incubation at 500 C for 30 min.; initial denaturation step of 940 C for 5 min. followed by 35 cycles of 940 C for 50 sec., 550 C for 1 min. and 680 C for 80 sec, followed by a final extension of 7 min at 680 C.
The PCR reactions were run on a 1.5% agarose gel to visualize the fragments amplified.
Real Time PCR – ZIKV (Realstar ZIKA Virus RT-PCR kit)
2-5 % of the samples of both Ae. aegypti and Ae. albopictus specimens distributed to all the Districts surveyed were processed by Real time PCR following Real star ZIKA RT-PCR kit, following their protocol.
30 µl (20 µl master mix +10 µl sample) reactions were setup in 96 well plates. Reporter Dye: used was FAM. Reaction parameters: 550 C for 20 minutes, 950 C for 2 minutes followed by 45 cycles of 950 C for 15 seconds, 550 C for 45 seconds, 720 C for 15 seconds and cooling cycle 370 C for 600 seconds.
Samples were observed as positive, where the CT values did not exceed 32 cycles.