Genetic characterization of Chikungunya virus from New Delhi reveal emergence of a new molecular signature in Indian isolates.

BACKGROUND: Chikungunya (CHIK) is currently endemic in South and Central India and exist as co-infections with dengue in Northern India. In 2010, New Delhi witnessed an outbreak of CHIK in the months October-December. This was the first incidence of a dominant CHIK outbreak in Delhi and prompted us to characterize the Delhi virus strains. We have also investigated the evolution of CHIK spread in India. FINDINGS: Clinical samples were subjected to RT-PCR to detect CHIK viral RNA. The PCR amplified products were sequenced and the resulting sequences were genetically analyzed. Phylogenetic analysis based on partial sequences of the structural proteins E1 and E2 revealed that the viruses in the latest outbreak exhibited ECSA lineage. Two novel mutations, E1 K211E and E2 V264A were observed in all Delhi isolates. In addition, CHIKV sequences from eight states in India were analyzed along with Delhi sequences to map the genetic diversity of CHIKV within the country. Estimates of average evolutionary divergence within states showed varying divergence among the sequences both within the states and between the states. We identified distinct molecular signatures of the different genotypes of CHIKV revealing emergence of a new signature in the New Delhi clade. Statistical analyses and construction of evolutionary path of the virus within the country revealed gradual spread of one specific strain all over the country. CONCLUSION: This study has identified unique mutations in the E1 and E2 genes and has revealed the presence of ancestral CHIKV population with maximum diversity circulating in Maharashtra. The study has further revealed the trend of CHIK spread in India since its first report in 1963 and its subsequent reappearance in 2005.

Dynamic expression of miRNAs across immature and adult stages of the malaria mosquito Anopheles stephensi.

BACKGROUND: MicroRNAs are small non-coding RNAs that are involved in various biological processes including insect development. Anopheles stephensi serves as primary vector of malaria parasite in Asia and exhibits holometabolous life cycle that involves four different stages of development. Regulation and role of mosquito miRNAs during various stages of mosquito development remain largely unknown. METHODS: High throughput small RNA sequencing was employed for identification and profiling of miRNAs across immature and adult stages of malaria vector, which were further validated using Northern hybridization and real time PCR. Target prediction and pathway analysis was carried out to understand the role of regulated miRNAs in insect development. Degradome sequencing was employed to identify cleaved targets of some regulated miRNAs. Loss of function strategy was employed for miR-989 to understand its probable role in female reproductive process. RESULTS: Small RNA sequencing and data analysis revealed 111 and 14 known and novel miRNAs respectively across all stages of Anopheles stephensi. Nine miRNAs showed gender specific regulation across different stages of mosquito development. Analysis of miRNAs revealed regulation of 24 and 26 miRNAs across different stages of male and female mosquito development respectively. mRNA targets and significant pathways targeted by regulated miRNAs were identified for each stage of mosquito development. Degradome sequencing revealed twenty nine cleaved targets of insect miRNAs. MicroRNA-989 showed significant up-regulation in the adult female as compared to adult male mosquito. Knockdown of miR-989 expression in adult female using miRNA specific antagomir affected targets playing roles in protein binding, proteolysis and nucleic acid binding in ovary tissue of female mosquito post blood feeding. CONCLUSIONS: This is the first comprehensive effort to understand regulation of Anopheles stephensi miRNAs across developmental stages of male and female mosquito. Preliminary role of regulated miRNAs in mosquito development was revealed by target prediction and pathway analysis. MicroRNA-989 emerged to have important roles in adult female mosquitoes showing significant up-regulation which was further studied using miR-989 specific antagomir. This study provides insights into mosquito development and reproductive process and has implications for effective control of mosquito population required for reducing spread of mosquito-borne infectious diseases.

Blood feeding and Plasmodium infection alters the miRNome of Anopheles stephensi.

Blood feeding is an integral process required for physiological functions and propagation of the malaria vector Anopheles. During blood feeding, presence of the malaria parasite, Plasmodium in the blood induces several host effector molecules including microRNAs which play important roles in the development and maturation of the parasite within the mosquito. The present study was undertaken to elucidate the dynamic expression of miRNAs during gonotrophic cycle and parasite development in Anopheles stephensi. Using next generation sequencing technology, we identified 126 miRNAs of which 17 were novel miRNAs. The miRNAs were further validated by northern hybridization and cloning. Blood feeding and parasitized blood feeding in the mosquitoes revealed regulation of 13 and 16 miRNAs respectively. Expression profiling of these miRNAs revealed that significant miRNAs were down-regulated upon parasitized blood feeding with a repertoire of miRNAs showing stage specific up-regulation. Expression profiles of significantly modulated miRNAs were further validated by real time PCR. Target prediction of regulated miRNAs revealed overlapping targeting by different miRNAs. These targets included several metabolic pathways including metabolic, redox homeostasis and protein processing machinery components. Our analysis revealed tight regulation of specific miRNAs post blood feeding and parasite infection in An. stephensi. Such regulated expression suggests possible role of these miRNAs during gonotrophic cycle in mosquito. Another set of miRNAs were also significantly regulated at 42 h and 5 days post infection indicating parasite stage-specific role of host miRNAs. This study will result in better understanding of the role of miRNAs during gonotrophic cycle and parasite development in mosquito and can probably facilitate in devising novel malaria control strategies at vector level.