Nation-wide vector surveillance did not indicate transmission of the “American lineage pandemic ZIKA virus” in India

Background In wake of the global health emergency declared by the World Health Organization (WHO) during 2016, on the outbreak of ZIKA pandemic, Indian Council of Medical Research (ICMR) carried out countrywide vector surveillance for ZIKA and DENGUE viruses (ZIKV & DENV) in India, as a preparedness measure. Methods The study incorporated high-risk zones distributed to 49 Districts in 14 states/ Union Territories (UT) of India during 2016-2019. Seven ICMR Institutions undertook the study, following a uniform Standard Operating Protocol. Aedes specimens sampled on weekly intervals were processed by multiplex Reverse transcriptase PCR for ZIKV/DENV and Real time RT-PCR of ZIKV, among few samples distributed to all the Districts. Results Altogether, 79492 specimens of Aedes mosquitoes in 6492 pools were processed for both ZIKV and DENV infections. Among these, three and 63 pools respectively were found positive for ZIKV and DENV. ZIKV infections were recorded from Aedes aegypti sampled during 2018 sporadic ZIKA outbreak in Jaipur, Rajasthan, which belonged to the Asian lineage, already circulating in the Country. Both Ae. aegypti and Aedes albopictus were found infected with DENV and were distributed to ten states/ UTs. Both male and female specimens of Ae. albopictus recorded DENV infections indicating trans-ovarial transmission of DENV in the species. Conclusion This national vector surveillance study evinced no active transmission of the “American lineage - pandemic ZIKA virus” in India during 2016-2019, although Asian lineage of the virus already circulating in the Country was detected from Ae. aegypti from Jaipur, Rajasthan.

Counties across the Globe (https://www.who.int/health-topics/zika-virus-disease#tab=tab_1) affecting a population of about 85000 [1]. In addition, the mutant strain of ZIKV (American Lineage) involved in this pandemic ZIKA caused incredible clinical complications such as microcephaly [2,3,4] and increase in the incidence of Gillian Barre Syndrome [5.6]. World Health Organization called for active surveillance in Countries where no outbreaks were reported initially, including India.
The primary route of transmission of ZIKV is through infected mosquito bites of Aedes mosquitoes such as Aedes aegypti [7,8,9] and Aedes albopictus [10,11]. In addition, other routes of transmission (congenital, sexual, transplacental and by contact with body uids) are also reported [12]. As a preparedness measure, Indian Council of  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 identi ed 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 4 o Celsius. These tubes were transported to the ICMR institutions once a week, where it was further processed for arboviral 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 identi cation. 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 4 o 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 deionized 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 ampli ed by performing a multiplex PCR [21,22].
The primers used are as follows:

Results
During the period of study, altogether 79492 specimens of Aedes mosquitoes were processed across the Country, 1470 specimens of Aedes vittatus. (Table 1). These were processed as 6492 pools for RT-PCR. Among these, three pools were found positive for ZIKV infection and 63 pools for DENV infection.
ZIKV infections were recorded from three pools of Ae. aegypti specimens collected during the outbreak investigation of ZIKA, which occurred in Jaipur, Rajasthan during 2018. On further analysis the ZIKV involved in the infections were found to be the Asian Lineage of the virus already circulating in the Country. The 1524 bp sequences Envelope gene ampli ed matched 100% with the human isolates collected from the same area from a parallel ICMR [23]. Rest of the Country did not report any infection of ZIKV in Aedes specimens sampled.
Dengue infections were recorded from both Ae. aegypti as well as from Ae. albopictus specimens, from Kerala (1) Besides, the infection status the study also provided an insight into the prevalence of these two major Aedes vector species in the Country. The state wise distribution of Aedes specimens samples are provided in  (Fig 2). Interestingly, natural infections with DENV was recorded in both Ae. aegypti as well as Ae. albopictus in the study. This study clearly evinced the role of both the species as major vectors of Dengue in the Country [24].
Real time RT-PCR was performed using Real star ZIKA Virus RT-PCR kit among 5% of pools distributed to all the   8425  18370  879  27674  2422  0  1  Puducherry  3225  749  302  4276  366  0  1  Tamil  Nadu   9881  882  0  10763  554  0  0   Total  21531  20001  1181  42713  3342  0  2  2  ICMR-NIMR,  New Delhi   Delhi  6736  0  0  6736  938  0  0  Gujarat  57  0  0  57  57  0  0  Rajasthan  246  0  0  246  65  3  0  Madhya  Pradesh   64  0  0  64  11  0  0   Total  7103  0  0  7103  1071  3  0  3 ICMR-NITM, Belagavi silently circulating in the Country. As the current pandemic strain is genetically related to Asian lineage, the study warrants a routine surveillance of the host as well as the vector species, so as prevent severe outbreaks similar to one that reported recently. We advocate a year round systematic surveillance program on emerging and re-emerging arbo-viral infections such as Zika, Dengue and Chikungunya in the human host as well as the vector species to tackle and prevent impending outbreaks of arbo-viral diseases in the Country.

Con ict of Interest Statement
The authors declare no con icts of interest.

Authors' contributions
Pradeep Kumar, N., the principal investigator of the multi-centric project planned and executed the study and wrote the manuscript with input from all authors. All other authors carried out their respective studies regionally and Distribution of study Districts, where Aedes samples were screened for ZIKV infection, Note: The designations employed and the presentation of the material on this map do not imply the expression of any opinion whatsoever on the part of Research Square concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. This map has been provided by the authors.