Jaspreet Jain

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.

Clinical, Serological, and Virological Analysis of 572 Chikungunya Patients From 2010 to 2013 in India

Background Chikungunya fever (CHIK) is a major public health concern in India. Characterized by acute fever with joint pain and swelling, most patients recover from this self-limiting illness in 7-10 days, with cessation of joint pain post-acute episode. However, in some patients, joint pain persists, lasting for months or even years. The precise correlates to the chronic phase of this debilitating illness and/or this remarkable heterogeneity in disease manifestation are poorly understood. Methods We evaluated 572 chikungunya patients from India who were recruited on the basis of positive real-time polymerase chain reaction and/or CHIK virus immunoglobulin (IgM) after receiving consent. Arthralgic conditions were monitored using visual analog score (VAS) 12 weeks after onset of fever in 130 patients. Initial viral load, IgG, and initial neutralization response were assayed and correlated with clinical and VAS information in 40 patients. Results Our extensive screening revealed that patients with higher initial viral loads during the acute phase of illness had poor prognosis at the post-acute phase with more restricted joint movement and higher VAS. Additionally, patients who showed early seroconversion to neutralizing IgG responses had better prognosis, as many of these patients did not manifest restricted joint movements at the post-acute phase. Conclusions Our study sheds light on chikungunya disease with respect to disease progression and assesses clinical, virological, and serological parameters of chikungunya disease severity. Importantly, it reveals that initial high viral load and neutralizing IgG response may function in a seemingly contrasting manner to negatively or positively dictate disease outcome.

Evidence for natural vertical transmission of chikungunya viruses in field populations of Aedes aegypti in Delhi and Haryana states in India-a preliminary report.

Aedes aegypti and Aedes albopictus are principal vectors for the transmission of chikungunya virus (CHIKV). India is a hub for both dengue and chikungunya infections and there are several reports of co-infection of dengue and chikungunya virus in the clinical scenario. The present pilot entomological survey was conducted to evaluate vertical transmission of CHIKV in Aedes field populations. Aedes immature (larvae and pupae) collection was done in 2012, over a period of six months from selected sites in Delhi and Haryana, India. The immatures collected were reared for adult emergence and species identification was done. A. aegypti male and female mosquitoes were separated and pooled collection spot-wise, RNA extracted and RT PCR performed to test for the presence of CHIKV in the pools. Container index (CI) and minimum infection rate (MIR) were estimated. From study areas that tested positive for CHIKV, adult collections were made and females upon feeding on uninfected blood in laboratory were allowed to lay eggs. The progeny that emerged from these field-collected mothers were tested for CHIKV presence. Our pilot survey showed the existence of A. aegypti population even during peak summer season in a few foci which eventually helped the mosquitoes to tide over adverse environmental conditions and with the start of rainfall, the population exploded within a short period of time. Immatures collected from field and progeny of adults collected from the field were CHIKV positive demonstrating the presence of vertical transmission of chikungunya virus in field population of A. aegypti. The present study further demonstrates the importance of identifying permanent breeding sites for proper Aedes species control.

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.

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.

In silico analysis of natural compounds targeting structural and nonstructural proteins of chikungunya virus

Background: Chikungunya fever presents as a high-grade fever during its acute febrile phase and can be prolonged for months as chronic arthritis in affected individuals. Currently, there are no effective drugs or vaccines against this virus. The present study was undertaken to evaluate protein-ligand interactions of all chikungunya virus (CHIKV) proteins with natural compounds from a MolBase library in order to identify potential inhibitors of CHIKV. Methods: Virtual screening of the natural compound library against four non-structural and five structural proteins of CHIKV was performed. Homology models of the viral proteins with unknown structures were created and energy minimized by molecular dynamic simulations. Molecular docking was performed to identify the potential inhibitors for CHIKV. The absorption, distribution, metabolism and excretion (ADME) toxicity parameters for the potential inhibitors were predicted for further prioritization of the compounds. Results: Our analysis predicted three compounds, Catechin-5-O-gallate, Rosmarinic acid and Arjungenin, to interact with CHIKV proteins; two (Catechin-5-O-gallate and Rosmarinic acid) with capsid protein, and one (Arjungenin) with the E3. Conclusion: The compounds identified show promise as potential antivirals, but further in vitro studies are required to test their efficacy against CHIKV.

Co-Habitation and Concurrent Infection of Dengue and Chikungunya Viruses in Aedes Aegypti Field Populations from India

Dengue and chikungunya have been identified as important re-emerging diseases in India. It has recently become a major health problem around the world, particularly in tropical and subtropical countries including India. Chikungunya fever is another re-emerging vector borne disease which is now being reported from areas previously unaffected with possibly changing epidemiology and severity of the disease. Aedes aegypti is the principal vector for the transmission of both of these arboviral infections. Information on vector population in the field vis-a-vis co-habitation of dengue and chikungunya viruses is of great importance in order to understand the role of vectors in the transmission of co-infections, but such information is presently lacking in India. We carried out a pilot survey in the states of Delhi and Haryana to estimate the presence of co-infections in Ae. aegypti during pre-monsoon, monsoon and post-monsoon seasons. This study is the first to report co-habitation of DENV and CHIKV in Ae.aegypti field population

Evaluation of an immunochromatography rapid diagnosis kit for detection of chikungunya virus antigen in India, a dengue-endemic country

BackgroundChikungunya virus (CHIKV) and dengue virus (DENV) are arboviruses that share the same Aedes mosquito vector, and there is much overlap in endemic areas. In India, co-infection with both viruses is often reported. Clinical manifestations of Chikungunya fever is often confused with dengue fever because clinical symptoms of both infections are similar. It is, therefore, difficult to differentiate from those of other febrile illnesses, especially dengue fever. We previously developed a CHIKV antigen detection immunochromatography (IC) rapid diagnosis kit [1]. The current study examined the efficacy of previously mentioned IC kit in India, a dengue-endemic country.MethodsSera from 104 CHIKV-positive (by qRT-PCR) and/or IgM-positive (ELISA) subjects collected in 2016, were examined. Fifteen samples from individuals with CHIKV-negative/DENV-positive and 4 samples from healthy individuals were also examined. Of the 104 CHIKV-positive sera, 20 were co-infected with DENV.ResultsThe sensitivity, specificity and overall agreement of the IC assay were 93.7, 95.5 and 94.3%, respectively, using qRT-PCR as a gold standard. Also, there was a strong, statistically significant positive correlation between the IC kit device score and the CHIKV RNA copy number. The IC kit detected CHIKV antigen even in DENV-co-infected patient sera and did not cross-react with DENV NS1-positive/CHIKV-negative samples.ConclusionsThe results suggest that the IC kit is useful for rapid diagnosis of CHIKV in endemic areas in which both CHIKV and DENV are circulating.

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.

In Vivo Evaluation of Withania somnifera–Based Indian Traditional Formulation (Amukkara Choornam), Against Chikungunya Virus–Induced Morbidity and Arthralgia

Chikungunya viral fever results in extreme morbidity and arthralgia in affected individuals. Currently, modern medicines providing symptomatic relief for the acute febrile phase and the chronic arthritic phase are only options available. Traditional Indian medical system, however, uses specific formulations for treatment of this infection; one such polyherbal formulation used to treat the postpyretic phase of chikungunya is amukkara choornam. The current study was undertaken to study the efficacy of amukkara choornam in the treatment of chikungunya in C57BL/6J mice. The formulation when administered to chikungunya-infected mice relieved morbidity and joint swelling. Analysis of virus clearance in brain and joint tissues on formulation treatment revealed a direct correlation of viral load in brain to morbidity during infection; likewise, joint swelling receded prior to complete viral clearance explaining possible immunomodulatory effect of amukkara choornam. This study provides insight into the possible mode of action of amukkara choornam during chikungunya.