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Zika virus positivity in Aedes aegypti mosquitoes and its genomic landscape from Pune, Western India
For correspondence: Dr Kavita Lole, Interactive Research School for Health Affairs, Bharti Vidyapeeth University, Pune 411 043, Maharashtra, India e-mail: lolekavita37@yahoo.com
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Received: ,
Accepted: ,
How to cite this article: Sreelakshmi PR, Raje M, Kumar S, Das T, Hari I, Jagtap M, et al. Zika virus positivity in Aedes aegypti mosquitoes and its genomic landscape from Pune, Western India. Indian J Med Res. 2026;164:287-94. doi: 10.25259/IJMR_3357_2025
Abstract
Background and objectives
Zika virus cases were confirmed from Pune, Western India, during an outbreak in June 2024. We conducted an entomological investigation in the Pune municipal corporation area and aimed to characterise the Zika virus from Aedes aegypti mosquitoes at the genomic level.
Methods
An entomological survey was conducted covering 16 locations in the Pune Municipal Corporation, between June and August 2024. Adult mosquitoes were collected using aspirators, while larvae were collected with the standard dipping and pipetting method. Aedes vector density was assessed, and samples were pooled. Zika virus positivity was assessed using real-time RT-qPCR. Genomic characterisation of the strains was achieved by whole-genome sequencing using the Oxford Nanopore technology platform.
Results
A high density of Aedes mosquitoes was revealed in the Pune area. Zika virus positivity was observed in 34/128 (26.5%) of Aedes aegypti mosquito pools. Full genome sequences of two ZIKV isolates were recovered from adult mosquito pools. Their time-scaled phylogenetic analysis showed grouping within the Asian lineage, having a recent ancestral time of evolution around 2014, indicating the introduction of newer virus strains in the recent past in India. Mutations E-V473M and motif E-VNDT, indicative of faster viral replication and higher mosquito infectivity, were noted in only one of the two isolates.
Interpretation and conclusions
With a high positivity of the Zika virus in Aedes aegypti, this study highlights the ongoing risk prevailing in the Pune municipal corporation area. Distinct characteristics of the circulating strains of the virus, and their evolutionary links to different global strains, underlines the importance of continuous genomic surveillance in vectors and hosts.
Keywords
Aedes aegypti
Entomological surveillance
Outbreak
Whole genome sequencing
Zika virus characterisation
The Zika virus is a mosquito-transmitted virus belonging to the genus Flavivirus and family Flaviviridae. The possible association of neurological complications like microcephaly among newborns and Guillain-Barré syndrome with Zika virus,1 led to the declaration of Zika virus infection as a public health emergency of international concern by the World Health Organization in the year 2016.2
Aedes mosquitoes have been incriminated as the principal vectors for transmitting the Zika virus,3 with Aedes aegypti being the primary vectors and other species as secondary vectors.4 High Aedes mosquito density, suitable climatic conditions, and a high proportion of naïve population for Zika virus favours outbreaks. In India, Zika virus outbreaks have occurred since 2017 in Rajasthan5,6 and thereafter in other States, including Gujarat, Tamil Nadu, Kerala, Karnataka, Maharashtra, and Uttar Pradesh.7 In this context, it is important to gain deeper insights into the transmission of the virus as well as to monitor any significant mutations of the virus in the vector. We report the entomological investigation undertaken during a Zika virus outbreak (June 2024) in Pune, and the characteristics of the virus isolated from Aedes aegypti mosquitoes at the genomic level.
Methods
This study was undertaken by the department of Entomology, ICMR-National Institute of Virology, Pune, Maharashtra, India.
Study setting
The study was carried out in the Pune municipal corporation region, the urban administrative area of Pune district, Maharashtra. Covering an area of 340 square km, the region caters to more than 33 lakh population8 under 16 wards. ICMR-National Institute of Virology confirmed the first Zika virus disease case from this region in June 2024. Following this, an entomological survey was carried out during July to August 2024 in the areas where Zika virus positive cases were reported ( Figure). The Pune municipal corporation’s map was generated using QGIS (Ver 3.4).
Entomological survey
Aedes adult mosquitoes and larvae were collected from the house of the reported case, as well as from the surrounding premises within a 400-meter radius. Oral and mechanical aspirators were used for adult mosquito collection, while larvae were collected from their habitats using glass or plastic pipettes and standard dipping methods. Aedes indices, including the house index (HI), container index (CI), and Breteau index (BI), were calculated. The per man-hr density (PMHD) was also computed for adult Aedes mosquitoes. After species-level identification using taxonomic keys, the mosquitoes were pooled according to locality, sex, and species. For adult pools, a maximum of 10 mosquitoes belonging to the same species, gender, and location were pooled together. Adult mosquitoes were homogenised mechanically. Gravid mosquitoes were processed only after the blood meal was digested. Fifty Culicinae larvae were grouped as one pool. Species identification of larvae was not done. The mosquito pools were further processed for Zika virus detection by real-time RT-PCR. Positive pools with a Ct value of less than 30 were selected for genome analysis.
Detection of Zika virus from field-caught mosquitoes
Real-time RT-PCR assay
Aedes aegypti mosquitoes, both adults and larvae, collected from different wards of Pune, were snap frozen in liquid nitrogen and homogenised using a chilled mortar-pestle in 1 mL chilled minimum essential medium (MEM) supplemented with 2% FBS (foetal bovine serum). Viral RNA was extracted using QIAamp viral RNA kit (Qiagen, Hilden, Germany) according to the manufacturer’s recommendations. RNA was eluted in 50 μL of nuclease-free water and stored at -80°C until use. Viral RNA copies were determined using real-time qRT-PCR using Zika virus forward primer- ZIKV 1086 (1086-1102) 5’-CCGCTGCCCAACACAAG-3’, reverse primer- 1162c (1162-1139) 5’-CCACTAACGTTCTTTTGCAGACAT-3’, and probe 5’-FAM AGCCTACCTTGACAAGCAGTCAGACACTCAA-3’.9 Ten-fold serial dilutions of Zika virus E gene RNA transcripts with known copy numbers (10 -107 copies/reaction) were evaluated in the same plate along with samples as an external standard. The thermal cycle conditions used for the current investigation were as follows: a single cycle of reverse transcription for 15 min at 48°C, held for 2 min at 95°C for reverse transcriptase inactivation and activation of DNA polymerase, followed by 40 cycles of amplification at 95°C for 15 sec and 60°C for 1 min. The amplification plot and the Ct-values were analysed using the appropriate software.
Whole-genome sequencing using the Oxford nanopore technology (ONT) platform
Whole-genome sequencing (WGS) of Zika virus from mosquitoes was done using the Oxford nanopore technology (ONT) MinION Mk1C device. Zika virus reference sequences were downloaded from the GenBank database, aligned, and used for designing primers for multiplex tiling PCR to cover the whole Zika virus genome, keeping a constant amplicon size of ∼1000 bp using Primal Scheme software10 ( https://primalscheme.com ). Library preparation, WGS, and analysis were carried out as described earlier.11
Sequence analysis
Data set
Complete/nearly complete (length > 9000 nt) genome sequences of Zika virus were downloaded from the NCBI virus portal12 by excluding sequences obtained from laboratory passaged or vaccine strains on 22 May 2025. From 1374 sequences, 1149 sequences (with human or mosquito as host) were selected for which both country and yr of collection were known. Further, the sequences having greater than 50 ambiguity characters ‘N’ were removed. Three sequences from the Pune 2024 outbreak [ZIKV NIV Homo sapiens IND 2024, PQ461653 (Aedes aegypti IND 2024), and PV893037 (ZIKV NIV Aedes aegypti IND 2024)] were then added to this dataset, and multiple sequence alignment was carried out using the MAFFT web server.13 Five sequences with multiple consecutive alignment gaps were further removed, resulting in the dataset of 824 sequences. To minimise redundancy, highly identical sequences (>99% nucleotide identity) were removed by using an in-house developed Python code, ensuring that representative sequences from each country and yr of collection are retained. It was also confirmed that all Indian/Malaysian strains and two top hits from the BLAST result of Indian strains were present in the final dataset. Thus, the final dataset used for further analysis included 123 Zika virus nucleotide sequences. Only coding sequences were used as the non-coding regions contained many alignment gaps.
Phylogeny
To evaluate the temporal signal, root-to-tip regression was performed in the software TempEst14 (v1.5.3, https://beast.community/tempest ) by locating the best-fit root position based on the neighbour-joining tree. A strong positive correlation (correlation coefficient = 0.92) between root-to-tip genetic distance and sampling yr for each tip was observed, indicating the presence of a temporal signal and suitability for molecular clock analysis. The best-suited nucleotide substitution model as identified by MEGA615 was ‘General Time Reversible’ with among-site rate heterogeneity and a significant proportion of invariable sites, viz. GTR + G +I. An evolutionary distance-scaled phylogenetic tree was constructed by the maximum likelihood algorithm at the IQ-TREE webserver with ultrafast bootstrap (UFBoot) analysis of 1000 iterations,16 for 123 nearly-complete Zika virus genomes from April 1947 to September 2021, downloaded from GenBank.
Molecular clock
To assess a time-scaled evolutionary relationship, Bayesian Markov Chain Monte Carlo (MCMC) analysis was performed as implemented in the BEAST 1.10.4 package.17 Three independent runs of the Markov chain, each with a length of 120 million and sampling frequency of 5000, were done. Two models of molecular clock, strict and uncorrelated relaxed lognormal, were fitted to the data set with a Bayesian skyline tree prior (which does not assume any pattern of population demography). The marginal likelihood values, estimated by path-sampling and stepping-stone methods, were compared to choose the best-fitted molecular clock model. The positive difference (>100) between the marginal likelihood of the lognormal clock and the strict clock, by both methods, strongly indicated that the relaxed lognormal clock fits better to the data. The convergence diagnostics of the MCMC analysis were done using Tracer 1.6. The effective sample size (ESS) for all parameters was greater than 200. Tree annotator was used to generate the maximum clade credibility (MCC) tree based on the combined trees of the three runs of the lognormal clock. FigTree v1.4.3 was used to visualise the MCC tree and to infer the tMRCA (time to most recent common ancestor) estimates for internal nodes of the tree.
Results
Vector indices
Aedes vector density was found to be high in the outbreak area. The house index, container index, and Breteau index were 30.2% (90/298), 37.9% (180/475), and 44.9% (134/298), respectively. Productive containers were obtained both indoors and outdoors. Outdoor non-discardable containers were the most productive breeding sites with a container index of 41.1% (130/316). Table I details the container preference of the Aedes mosquitoes from the study area. The average Per man-hr density (PMHD) of adult mosquitoes was 6.88. The PMHD during the study period ranged between 3.5 and 10.5. The species-wise average density was less for Aedes albopictus (1.6) as compared to Aedes aegypti (10.8).
| Location | Container category | Type of container | Number of positive containers, container index (%) |
|---|---|---|---|
| Indoor | Non-discardable | Refrigerator, air-cooler, earthen pot, indoor plant pots, small metal drums | 12, 20 |
| Outdoor | Non-discardable | Plastic drums, cement tanks, metal drums | 130, 41.1 |
| Discardable | Plastic sheets, tyres, plastic containers | 38, 37.6 |
ZIKV RNA positivity in mosquitoes
A total of 577 adult mosquitoes and 3190 larvae were collected and sorted into 128 pools based on species and locations. Out of 128 pools, 54 comprised of adult mosquitoes and 74 were larval pools. Forty-six pools were of adult Aedes aegypti mosquitoes (9 male and 37 female pools), and 8 pools were of adult Aedes albopictus mosquitoes (4 male and 4 female pools). Of the total 128 pools, 34 (26.5%) tested positive for Zika virus RNA in real-time RT-PCR. The average Ct value for the mosquito pools was 35.98. Of 34 positive pools, 29 were adult Aedes aegypti, 4 were larval, and 1 was an Aedes albopictus pool. Two adult Aedes aegypti pools belonging towards the Erandwane and Yewalewadi in the Pune Municipal Corporation area had Ct values below 30 (22 and 21, respectively). Table II gives the details of the mosquito pools from various locations within the study area. The two Zika virus-positive mosquito samples showing higher viral load were processed for virus whole genome sequence analysis to understand the genetic composition of the circulating strain/s.
| Location | Total number of pools made | Number of positive pools |
|---|---|---|
| Aundh | 5 | 2 |
| Kothrud | 3 | 0 |
| Warje | 25 | 7 |
| Hadapsar | 8 | 1 |
| Keshav Nagar | 10 | 0 |
| Nagar road/Yerwada | 14 | 3 |
| Sangamwadi | 10 | 1 |
| Tilak road | 10 | 3 |
| Bhawani Peth | 8 | 1 |
| Bibwewadi | 9 | 3 |
| Dhankawdi | 17 | 6 |
| Ghole road | 2 | 2 |
| Dhole Patil road | 2 | 2 |
| Vishrambaug Wada | 1 | 0 |
| Kondhawa, Yewale wadi | 3 | 3 |
| Wanowrie | 1 | 0 |
| Total | 128 | 34 |
Whole genome sequence analysis
The amplification scheme allowed 98% recovery of the Zika virus genome from the first pool (isolate 1) and ∼95% genome recovery from the second pool (isolate 2). Isolates 1 and 2 showed a significant difference at nucleotide as well as at amino acid levels, as shown in Tables III and Supplementary Table I.
| Genomic region | Amino acid position | Amino acid in Malaysia 1966 isolate KX601167 | Nucleotide | Amino acid | |
|---|---|---|---|---|---|
| Aedes aegypti Pune 2024 isolate 1 (PQ461653) | Aedes aegypti Pune 2024 isolate 2 (PV893037) | ||||
| PreM | 9 | A | C499T | A | V |
| E | 153 | V | GTT 1434-36 | V | DEL |
| 154 | N | AAT 1437-39 | N | DEL | |
| 155 | D | GAC 1440-42 | D | DEL | |
| 156 | T | ACA 1443-45 | T | DEL | |
| 157 | G | GGA 1446-48 | G | DEL | |
| 324 | G | G 1947 T | G | W | |
| 419 | K | A2233G | R | K | |
| 470 | T | A2385G | T | A | |
| 473 | V | G2394 | M (G to A) | L (G to T) | |
| NS2A | 143 | A | C3973T | V | V |
| NS2B | 102 | V | T4528C | V | A |
| NS3 | 122 | D | C4979G | E | D |
| 215 | T | A5256G | T | A | |
| 314 | F | T5553C and C5555T | F | L | |
| NS4B | 246 | G | G7650A | G | S |
| NS5 | 204 | G | G8278DEL | DEL | G |
| 495 | E | G9150C | E | Q | |
| 526 | T | C9244T | I | I | |
| 530 | K | A9256G | R | R | |
| 560 | K | A9346G | K | R | |
| 561 | G | G9349A | G | E | |
| 633 | M | A9564G | M | V | |
| 672 | I | A9681G | I | V | |
PreM, pre membrane; E, envelope region; NS2A, non-structural region 2A; NS2B, non-structural region 2B; NS3, non-structural region 3; NS4B, non-structural region 4B; NS5, non-structural region 5
The Zika virus phylogenetic tree outlined two distinct primary lineages, the African and the Asian lineage (Supplementary Fig. 1A). The two Pune 2024 Aedes aegypti ZIKV sequences clustered within the Asian lineage viruses. Isolate 1 sequence (PQ461653) clustered along with the recently reported Zika virus sequence from a human blood sample collected during the Pune 2024 outbreak, while the Isolate 2 sequence (PV893037) clustered on a separate branch.
The evolutionary timescale of Indian Zika virus strains is shown in Supplementary Figure 1B. The overall mean rate of evolution was found to be 7.4 x 10-4 substitutions (subs)/site/yr with 95% highest posterior density (HPD) limits 6.0, 8.7 x 10-4 subs/site/yr, while the mean root age was found to be 160.6 yr (95% HPD: 111.8, 214.1), indicating the likely existence of the Zika virus since 1863 (1810, 1912). The Pune 2024 outbreaks formed a single phylogenetic group that also included other Zika virus strains from India (nine sequences), and sequences reported from Kenya (2024) and Seychelles (2024). The tMRCA of this group was estimated to be around 2014 (HPD: 2012, 2015). The members of this phylogenetic group showed percent nucleotide identity (PNI) of 98.9%. This group showed further division into two subgroups, A and B. One of the two Pune 2024 isolates (PV893037) formed subgroup A along with 7 Indian and 2 Kenyan sequences with a PNI of 99.1%. The second isolate from this outbreak (PQ461653) and two Seychelles sequences with a PNI of 98.97% formed the subgroup B.% nucleotide divergence between A and B was 1.38%.
Amino acid changes in the structural and non-structural proteins in Pune 2024 strains of Zika virus
We analysed the amino acid changes in Pune 2024 strains and compared them with other Indian, Malaysian, South American (Brazil), and closely related (Kenyan and Seychelles) strains (Supplementary Table). The mosquito isolate-1 (PQ461653) from this outbreak showed the presence of a VNDT sequence motif at positions 153-156 (443-446 in polyprotein) in the E surface glycoprotein while, while the Pune 2024 mosquito isolate-2 (PV893037) showed deletion of this motif. Isolate-2 showed the presence of the V473L mutation in the E protein, while the isolate-1 showed the V473M mutation. The mosquito isolate-1 also harboured an M872V mutation in the polymerase domain of the NS5 protein. The presence of this mutation in the mosquito isolate-2 could not be confirmed due to the absence of sequence from this region. The mutations distinguishing the Pune 2024 mosquito isolates, which made them belong to the two different subgroups A and B are given in Table III.
Discussion
During the period June- September 2024, Pune city confirmed an outbreak with 108 cases of Zika virus, including 45 antenatal women.18 The entomological investigation carried out during this period revealed high larval indices with a house index of 30.2%, container index of 37.89%, and breteau index of 44.9%. Zika virus RNA was positive among 26.5% of the mosquito pools. The near-complete genome of the Zika virus was retrieved from two pools, and phylogenetic analysis, time-scaled evolutionary analysis, and mutational analysis studies were undertaken.
Zika virus positivity from mosquito pools has been reported previously in India. Akthar et al19 have reported Zika virus positivity in field-caught, mosquitoes during virus surveillance studies in the vectors conducted between 2016 and 2021. During the same period, a national-level surveillance study conducted by the Indian Council of Medical Research for the Zika virus and Dengue virus in Aedes mosquitoes also revealed the presence of the Zika virus in mosquito pools.20 Mosquito pools tested during the Zika virus outbreak in Rajasthan demonstrated positivity of 5.45%.20 Recently, the Zika virus was detected in Aedes mosquito pools from Karnataka.21
The phylogenetic tree showed that the majority of the recent Indian Zika virus strains evolved as a separate cluster within the Asian lineage. These Indian strains retained close phylogenetic relationships with Kenya (2024), and Seychelles (2024) virus strains. Interestingly, the single Zika virus strain isolated from a sporadic human Zika case in 2021 from Pune (OK054351) showed phylogenetic relationship with Malaysian 1966 strains (KX377336, HQ234499, and KX601167). These Malaysian isolates are the earliest known strains of the Asian lineage. This isolate appeared to share a common ancestor with that of the Malaysian lineage, with an estimated tMRCA of around 1959 (95% HPD: 1952, 1964).
The tMRCA for the phylogenetic group formed by the majority of the recent Indian Zika virus strains associated with outbreaks in India was estimated to be around 2014. This indicates the introduction of Zika virus strains in the recent past in India. An earlier report on detection of Zika virus neutralising antibodies in the sera from Indian residents suggests that Zika virus existed in India, possibly since 1947.22 This provides evidence that Zika virus was in widespread circulation silently, causing mostly asymptomatic or mild infections in the Southeast Asia Region.23 The identification of several distinct mutations in these sequences, with ancestry (nodes A and B) at similar time frames, could possibly indicate co-evolution of independent strains in the same geographical location.
The mutations distinguishing the Pune 2024 mosquito isolates in view of the amino acids noted in previous Indian, other closely related strains, and important virulent strains, were analysed. The E- V473M mutation, in the majority of the current Zika virus strains in India, possibly has translated into greater peripheral viraemia and efficient mosquito infection and spread.24 Noted as a unique mutation in other recent Indian outbreaks, the EV473L mutation was also observed in one of the current isolates (PV893037).25 The significance of this mutation is yet to be established. It was noted that this mutation co-occurs with additional mutations, D348N and T470A, in the E protein. The isolate PV893037 showed the presence of T470A. Further, the motif E-VNDT that incorporates an N-linked glycosylation site and is known as a determinant of Zika virus virulence,26 was not noted in the same isolate (PV893037). The motif has been noted widely in isolates from recent outbreaks also.26
The Pune 2024 strains were significantly different from Zika virus strains from South America that were responsible for large-scale outbreaks in 2015-2017. Mutations associated with neurovirulence and microcephaly, including the S139N prM mutation27 and T233A in NS1 protein28 were absent in both Pune 2024 strains.
This study adopted an integrated approach of an entomological survey along with in-depth analysis of the genomic data identified Zika virus strains. The extensive ward-level entomological surveillance undertaken in this study could have enhanced the likelihood of detecting infected vectors, resulting in a higher percentage positivity. The study has limitations. Despite identifying Zika virus positivity in 34 of the 128 mosquito pools, full-length genome sequences could be obtained from only two pools due to high Ct values in the remaining positive samples. Some false positivity at high Ct values is likely, as these are close to the limit of detection of the assay.
The findings of high vector density and high percentage positivity of the Zika virus in the vectors are an indicator of the possible risk of future Zika virus outbreaks in the region and call for strengthened surveillance. Further monitoring of the divergence of Zika virus groups noted in this study needs to be continued to track the independent evolution of circulating strains. Though mutations associated with neuro-virulence are absent in the current Indian strains, the presence of other mutations warrants constant monitoring of emerging viruses and their pathogenesis in the future.
Author contributions
SPR: Planning of entomological investigation, collection and interpretation of data, manuscript writing; MR: Molecular detection of ZIKV in mosquito samples, manuscript writing; SK: Molecular detection of ZIKV in mosquito samples, manuscript writing; TD: Molecular detection of ZIKV in mosquito samples, manuscript writing; IH: Outbreak investigation, entomological survey and vector indices calculation; MJ: Deployment of State insect collectors team for entomological survey; AR, SH: Whole genome sequencing and phylogenetic tree analysis, analysis of amino acid changes; SC, AW: Evolutionary analysis, manuscript writing; SD, PS, VI: Entomological survey, insect identification and pool preparation, coordination with the State entomology team; GS: Identification of sites with positive ZIKV cases, intellectual input, manuscript writing; KL: Conception and designing of the study, genome data analysis and interpretation, manuscript writing. All authors have read and approved the final printed version of the manuscript.
Financial support and sponsorship
The study received funding support from the ICMR-National Institute of Virology’s institutional fund.
Conflicts of Interest
None.
Use of Artificial Intelligence (AI)-Assisted Technology for manuscript preparation
The authors confirm that there was no use of AI-assisted technology for assisting in the writing of the manuscript and no images were manipulated using AI.
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