Determination of Species Identity and Genetic Diversity of Aedes aegypti and Other Medically Important Mosquitoes of India Using DNA Barcoding.

Aedes aegypti-borne viruses (i.e., dengue, chikungunya, and Zika) have become endemic to India, posing a severe threat to public health. Vector control remains the mainstay of disease management due to nonavailability of licensed vaccines/therapeutics. Conventional morpho-taxonomical methods cannot differentiate between closely related sibling species or species complexes, and hence we evaluated two molecular markers, mitochondrial cytochrome c oxidase subunit 1 (Cox1) and nuclear DNA internal transcribed spacer 2 (-2) gene sequences, to characterize seven populations of Ae. aegypti and four medically important mosquito species (Aedes albopictus, Anopheles stephensi, Culex tritaeniorhyncus, and Culex murrelli). DNA extracted from the 11 mosquito populations (two mosquitoes per population) was polymerase chain reaction amplified, sequenced, and analyzed. Molecular characterization was found to be congruent with morphological identification, suggesting no variants or cryptic species exist in Ae. aegypti and the other mosquitoes studied. Phylogenetic analysis with sequences obtained with Cox1 gene of Ae. aegypti and other Aedes and non-Aedes mosquito species showed clustering of sequences from different species representing different clades, distinctly separating one taxon from the other, whereas ITS-2 sequences of Aedes aegypti from across the world clustered tightly. Nucleotide divergence values revealed a low percentage of intraspecies variation and a higher percentage of interspecies variation. The present study authenticates the applicability of Cox1 and ITS-2 in the precise identification of Ae. aegypti mosquitoes against cryptic or sibling species. Cox1 appeared to be a more reliable marker because it showed distinct clustering of mosquito species, and some sequence variations to represent genetic diversity.

Metagenomic Analysis of Viromes of Aedes Mosquitoes across India.

Metagenomic analysis of Aedes aegypti and Ae. albopictus mosquitoes from diverse geographical regions of India revealed the presence of several insect viruses of human interest. Most abundant reads found in Ae. aegypti mosquitoes were of Phasi Charoen-like virus (PCLV), Choristoneura fumiferana granulovirus (CfGV), Cell fusing agent virus (CFAV), and Wenzhou sobemo-like virus 4 (WSLV4), whereas WSLV4 and CfGV constituted the highest percentage of reads in Ae. albopictus viromes. Other reads that were of low percentage included Hubei mosquito virus 2 (HMV2), Porcine astrovirus 4 (PAstV4), and Wild Boar astrovirus (WBAstV). PCLV and CFAV, which were found to be abundant in Ae. aegypti viromes were absent in Ae. albopictus viromes. Among the viromes analyzed, Ae. aegypti sampled from Pune showed the highest percentage (79.82%) of viral reads, while Ae. aegypti mosquitoes sampled from Dibrugarh showed the lowest percentage (3.47%). Shamonda orthobunyavirus (SHAV), African swine fever virus (ASFV), Aroa virus (AROAV), and Ilheus virus (ILHV), having the potential to infect vertebrates, including humans, were also detected in both mosquito species, albeit with low read numbers. Reads of gemykibivirus, avian retrovirus, bacteriophages, herpesviruses, and viruses infecting protozoans, algae, etc., were also detected in the mosquitoes. A high percentage of reads in the Ae. albopictus mosquito samples belonged to unclassified viruses and warrant further investigation. The data generated in the present work may not only lead to studies to explain the influence of these viruses on the replication and transmission of viruses of clinical importance but also to find applications as biocontrol agents against pathogenic viruses.

Genomic Characterization of Mosquito Isolates of Chikungunya Virus (Outbreak Strains 2022) Using Next-Generation Sequencing.

Background: A massive outbreak of dengue-like illness was reported from Pune district of Maharashtra, India during May-June 2022. Isolation and characterization of the etiological agent at genomic level for possible mutations that led to higher transmissibility is the topic of the study. Methods: Entomological investigations were carried out by ICMR-National Institute of Virology (Pune, India); Aedes aegypti mosquitoes were collected and processed for virus detection by molecular techniques. Positive mosquito pools were processed for virus isolation in cell culture. Sanger sequencing and whole-genome sequencing (WGS) using Oxford Nanopore Technology platform were used for genomic characterization. Results: Reverse transcriptase RT-PCR and qRT-PCR analysis detected chikungunya virus (CHIKV) in mosquito samples. Six CHIKV isolates were obtained. WGS revealed four nonsynonymous mutations in the structural polyprotein region, and five in the nonstructural polyprotein encoding region when compared with Yawat-2000 and Shivane-2016 strains. Sixty-four nucleotide changes in the nonstructural polyprotein region and 35 in the structural polyprotein region were detected. One isolate had an exclusive amino acid change, T1123I, in the nsP2 (protease) region. Conclusion: Abundant Ae. aegypti breeding and detection of CHIKV RNA in mosquitoes confirmed it as a chikungunya outbreak. Novel mutations detected in the epidemic strain warrants investigations to address their role in disease severity, transmission, and fitness.

Abundance of Phasi-Charoen-like virus in Aedes aegypti mosquito populations in different states of India.

Mosquitoes are known to harbor a large number of insect specific viruses (ISV) in addition to viruses of public health importance. These ISVs are highly species specific and are non-pathogenic to humans or domestic animals. However, there is a potential threat of these ISVs evolving into human pathogens by genome alterations. Some ISVs are known to modulate replication of pathogenic viruses by altering the susceptibility of vector mosquitoes to pathogenic viruses, thereby either inhibiting or enhancing transmission of the latter. In the present study, we report predominance of Phasi Charoen-like virus (PCLV, Family: Phenuviridae) contributing to >60% of the total reads in Aedes aegypti mosquitoes collected from Pune district of Maharashtra state using next generation sequencing based metagenomic analysis of viromes. Similar results were also obtained with mosquitoes from Assam, Tamil Nadu and Karnataka states of India. Comparison of Pune mosquito sequences with PCLV Rio (Brazil) isolate showed 98.90%, 99.027% and 98.88% homologies in the S, M and L segments respectively indicating less genetic heterogeneity of PCLV. The study also demonstrated occurrence of transovarial transmission as seen by detection of PCLV in eggs, larvae, pupae and male mosquitoes. Ae. aegypti mosquitoes collected from Pune also showed a large number of reads for viruses belonging to Baculoviridae, Rhabdoviridae, Genomoviridae and Bunyaviridae families. The role of PCLV in the replication of dengue and chikungunya virus is yet not clear. It warrants further studies to know the significance of PCLV and other ISVs on the replication and transmission of Ae. aegypti borne pathogenic viruses, especially in the absence of prophylactics or therapeutics.