Jatin Shrinet

Next Generation Sequencing Reveals Regulation of Distinct Aedes microRNAs during Chikungunya Virus Development

Background Application of genomics and Next Generation sequencing has led to the identification of new class of cellular functional molecules, namely, small RNAs. Of the several classes of ncRNAs (non-coding RNA), microRNAs have been demonstrated to exert determinative influence on various cellular processes. It is becoming abundantly clear that host/vector/pathogen encoded microRNAs impact eventual pathogenesis. In this context, the participation of vector based microRNAs in disease transmission and pathogen development is being investigated intensively. A few studies have highlighted the role of vector encoded microRNAs in pathogen infection. We conducted this study to evaluate the role of host miRNAs upon CHIKV (Chikungunya Virus) infection in an important vector, Aedes albopictus. Findings We identified 88 and 79 known miRNAs in uninfected and CHIKV infected Ae. albopictus Singhs cell line respectively. We further identified nine novel miRNAs in Ae. albopictus. Comparison of the two libraries revealed differential expression of 77 common miRNAs between them. CHIKV infection specifically altered the miRNA profile of a specific set of eight miRNAs. Putative targets of these regulated miRNAs were identified and classified into their pathways. Conclusions In our study we have identified and described the profiles of various miRNAs upon CHIKV infection in Ae. albopictus. This investigation provides an insight about cellular modification by miRNAs during CHIKV infection and the results provide leads for identifying potential candidates for vector based antiviral strategies.

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

BackgroundChikungunya (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.FindingsClinical 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.ConclusionThis 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.

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.

Serum metabolomics analysis of patients with chikungunya and dengue mono/co-infections reveals distinct metabolite signatures in the three disease conditions.

Chikungunya and dengue are arboviral infections with overlapping clinical symptoms. A subset of chikungunya infection occurs also as co-infections with dengue, resulting in complications during diagnosis and patient management. The present study was undertaken to identify the global metabolome of patient sera infected with chikungunya as mono infections and with dengue as co-infections. Using nuclear magnetic resonance (NMR) spectroscopy, the metabolome of sera of three disease conditions, namely, chikungunya and dengue as mono-infections and when co-infected were ascertained and compared with healthy individuals. Further, the cohorts were analyzed on the basis of age, onset of fever and joint involvement. Here we show that many metabolites in the serum are significantly differentially regulated during chikungunya mono-infection as well as during chikungunya co-infection with dengue. We observed that glycine, serine, threonine, galactose and pyrimidine metabolisms are the most perturbed pathways in both mono and co-infection conditions. The affected pathways in our study correlate well with the clinical manifestation like fever, inflammation, energy deprivation and joint pain during the infections. These results may serve as a starting point for validations and identification of distinct biomolecules that could be exploited as biomarker candidates thereby helping in better patient management.

Dengue Chikungunya co-infection: A live-in relationship??

Dengue and Chikungunya are viral infections that are a major public health hazard in recent times. Both these infections are caused by RNA viruses termed arboviruses owing to their requirement of an arthropod vector to get transmitted to vertebrate hosts. Apart from sharing a common vector, namely Aedes mosquitoes, these infections are also characterized by overlapping clinical presentations and are known to exist as co-infection. The present review traces the history and evolution of co-infection across the globe and provides specific compilation of the scenario in India. Furthermore, clinical manifestations during co-infection are discussed. Lastly, up-to-date information with respect to vector behaviour during co-infection both under laboratory conditions and in natural Aedes populations is reviewed.

Inference of the oxidative stress network in Anopheles stephensi upon Plasmodium infection.

Ookinete invasion of Anopheles midgut is a critical step for malaria transmission; the parasite numbers drop drastically and practically reach a minimum during the parasites whole life cycle. At this stage, the parasite as well as the vector undergoes immense oxidative stress. Thereafter, the vector undergoes oxidative stress at different time points as the parasite invades its tissues during the parasite development. The present study was undertaken to reconstruct the network of differentially expressed genes involved in oxidative stress in Anopheles stephensi during Plasmodium development and maturation in the midgut. Using high throughput next generation sequencing methods, we generated the transcriptome of the An. stephensi midgut during Plasmodium vinckei petteri oocyst invasion of the midgut epithelium. Further, we utilized large datasets available on public domain on Anopheles during Plasmodium ookinete invasion and Drosophila datasets and arrived upon clusters of genes that may play a role in oxidative stress. Finally, we used support vector machines for the functional prediction of the un-annotated genes of An. stephensi. Integrating the results from all the different data analyses, we identified a total of 516 genes that were involved in oxidative stress in An. stephensi during Plasmodium development. The significantly regulated genes were further extracted from this gene cluster and used to infer an oxidative stress network of An. stephensi. Using system biology approaches, we have been able to ascertain the role of several putative genes in An. stephensi with respect to oxidative stress. Further experimental validations of these genes are underway.

Analysis of coevolution in nonstructural proteins of chikungunya virus.

BackgroundRNA viruses are characterized by high rate of mutations mainly due to the lack of proofreading repair activities associated with its RNA-dependent RNA-polymerase (RdRp). In case of arboviruses, this phenomenon has lead to the existence of mixed population of genomic variants within the host called quasi-species. The stability of strains within the quasi-species lies on mutations that are positively selected which in turn depend on whether these mutations are beneficial in either or both hosts. Coevolution of amino acids (aa) is one phenomenon that leads to establishment of favorable traits in viruses and leading to their fitness.ResultsFourteen CHIKV clinical samples collected over three years were subjected to RT-PCR, the four non-structural genes amplified and subjected to various genetic analyses. Coevolution analysis showed 30 aa pairs coevolving in nsP1, 23 aa pairs coevolving in nsP2, 239 in nsP3 and 46 aa coevolving pairs in nsP4 when each non-structural protein was considered independently. Further analysis showed that 705 amino acids pairs of the non-structural polyproteins coevolved together with a correlation coefficient of ≥0.5. Functional relevance of these coevolving amino acids in all the nonstructural proteins of CHIKV were predicted using Eukaryotic Linear Motifs (ELMs) of human.ConclusionsThe present study was undertaken to study co-evolving amino acids in the non-structural proteins of chikungunya virus (CHIKV), an important arbovirus. It was observed that several amino acids residues were coevolving and shared common functions.

Aedes aegypti microRNA miR-2b regulates ubiquitin-related modifier to control chikungunya virus replication

Arboviruses that replicate in mosquitoes activate innate immune response within mosquitoes. Regulatory non-coding microRNAs (miRNA) are known to be modulated in mosquitoes during chikungunya infection. However, information about targets of these miRNAs is scant. The present study was aimed to identify and analyze targets of miRNAs that are regulated during chikungunya virus (CHIKV) replication in Aedes aegypti cells and in the mosquito. Employing next-generation sequencing technologies, we identified a total of 126 miRNAs from the Ae. aegypti cell line Aag2. Of these, 13 miRNAs were found to be regulated during CHIKV infection. Putative targets of three of the most significantly regulated miRNAs- miR-100, miR-2b and miR-989 were also analyzed using quantitative PCRs, in cell lines and in mosquitoes, to validate whether they were the targets of the miRNAs. Our study expanded the list of miRNAs known in Ae. aegypti and predicted targets for the significantly regulated miRNAs. Further analysis of some of these targets revealed that ubiquitin-related modifier is a target of miRNA miR-2b and plays a significant role in chikungunya replication.

Understanding Oxidative Stress in Aedes during Chikungunya and Dengue Virus Infections Using Integromics Analysis

Arboviral infection causes dysregulation of cascade of events involving numerous biomolecules affecting fitness of mosquito to combat virus. In response of the viral infection mosquito’s defense mechanism get initiated. Oxidative stress is among the first host responses triggered by the vector. Significant number of information is available showing changes in the transcripts and/or proteins upon Chikungunya virus and Dengue virus mono-infections and as co-infections. In the present study, we collected different -omics data available in the public database along with the data generated in our laboratory related to mono-infections or co-infections of these viruses. We analyzed the data and classified them into their respective pathways to study the role of oxidative stress in combating arboviral infection in Aedes mosquito. The analysis revealed that the oxidative stress related pathways functions in harmonized manner.

Differential Proteome Analysis of Chikungunya Virus and Dengue Virus Coinfection in Aedes Mosquitoes

Chikungunya virus (CHIKV) and dengue virus (DENV) are important arboviruses transmitted by Aedes mosquitoes. These viruses are known to coexist within the same vector and coinfect the same host. Although information is available on the mechanism of replication of CHIKV and DENV when present independently in a vector, reports are lacking on the dynamics of virus-vector interactions when these viruses coexist in a mosquito. The current study attempts to understand the perturbations in the proteome of Aedes mosquitoes when infected with CHIKV and DENV either independently or together. Global proteome profiling of chikungunya and dengue mono- and coinfection revealed 28 proteins to be significantly regulated. Validation of the transcripts of these proteins using qRT-PCR indicated differences in the expression patterns between transcript profiling and quantitative proteome analyses. Pathway analysis of the 28 differentially regulated proteins revealed 11 significant pathways, which include oxidative phosphorylation, carbon metabolism, and glycolysis/gluconeogenesis.