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.

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.

Rainfall and Chikungunya incidences in India during 2010-2014.

Changing climate scenario has resulted in recent emergence and re-emergence of various arboviral diseases including Chikungunya. This disease is caused by Chikungunya virus (CHIKV), which belongs to Togaviridae family of viruses and spread by Aedes mosquitoes. A resurgence of CHIKV and its rapid global spread has been observed since 2004. The disease reemerged in India in 2005, after a gap of 32 years, causing massive outbreaks in some states and circulating thereafter. In the present paper we analyze CHIKV incidence data from India (2010-2014) with a view to understand association with environmental parameters, if any. Data on country-wide occurrences of CHIKV cases were considered from the National Vector Borne Disease Control Board, India. Meteorological data for different climatic subdivisions of India were obtained and processed mathematically. State-wise association of number of cases with rainfall, if any, were studied by statistical analyses. We observe that prevailing temperature range was favorable for CHIKV propagation and the occurrences were modulated by average rainfall. Most affected states were West Bengal, Maharashtra and Karnataka. Overall for India, favorable climatic conditions have contributed to incidences of CHIKV during the study period. There is strong positive association between rainfall variations and occurrence of CHIKV cases.

Outbreak of chikungunya fever, Dakshina Kannada District, South India, 2008.

The outbreak of chikungunya fever that surfaced in India during late 2005 has affected more than 1.56 million people, spread to more than 17 states/union territories, and is still ongoing. Many of these areas are dengue- and leptospirosis-endemic settings. We carried out a cross-sectional survey in one such chikungunya-affected location in Dakshina Kannada District of Karnataka State to estimate the magnitude of the epidemic and the proportion of chikungunya virus (CHIKV) infections that remained clinically inapparent. The seropositivity for CHIKV infection was 62.2%, and the attack rate of confirmed CHIK fever was 58.3%. The proportion of inapparent CHIKV infection was 6.3%. The increasing trend in the seropositivity and attack rate of CHIKV infection with age group was statistically significant. The present study is an indicator of the magnitude of the ongoing outbreak of CHIKV infection in India that started during 2005-2006.