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53 result(s) for "Ray, Pratima"
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Virus load and clinical features during the acute phase of Chikungunya infection in children
Chikungunya virus (CHIKV) infection is a long known mosquito-borne disease that is associated with severe morbidity, characterized by fever, headache, rashes, joint pain, and myalgia. It is believed that virus load has relation with severity of clinical features. We performed this study to assess the relationship between virus load and clinical features in children during the acute phase of CHIKV infection, in order to draw insights for better-informed treatment. Between June 1, 2009, and May 31, 2010, 338 patients with fever and susceptive to CHIKV during first 4 days of illness were prospectively enrolled from Karnataka Institute of Medical Sciences, Hubli in our hospital based cross sectional observational study. Sybr green quantitative reverse transcription polymerase chain reaction was performed to estimate the virus load. Quantitative RT-PCR was positive for CHIKV in 54 patients. The median copy number of CHIKV was 1.3x 108 copies/ml (1.7x105-9.9x109 copies/ml). Among the observed clinical features, a statistically significant difference in log mean virus load was found between patients with and without myalgia (log mean 7.50 vs 8.34, P = 0.01). Patients with myalgia had lower virus load and those without myalgia had a higher virus load.
Combined miRNA and mRNA Signature Identifies Key Molecular Players and Pathways Involved in Chikungunya Virus Infection in Human Cells
Since its discovery, Chikungunya fever caused by a virus (CHIKV) has ravaged most of Africa and Southeast Asia. Despite there being more than a million reported cases in India alone and the seriousness of the disease in the chronic phase, a clear understanding of the disease pathogenesis and host response remains elusive. Here, we use microarray technology and quantitative PCR method to establish the complete miRNA, snoRNA and mRNA signature of host response upon CHIKV infection in human cell line infection model, HEK293T. The results were further validated in human primary cells (dermal fibroblasts). miRNA expression profiling revealed regulation of 152 miRNAs post CHIKV infection. An interesting overlap in miRNA signature was seen majorly with HCV, HPV and HIV1 virus. The microarray data further validated by qRT-PCR revealed induction of miR-744, miR-638, miR-503 and others among the top upregulated miRNAs. Notably, we found induction of snoRNAs belonging to C/D cluster including close paralogs of U3, U44, U76 and U78 snoRNAs. Genes were found to be differentially expressed along 3 major pathways; TGF-β, endocytosis and the cell cycle pathways. qRT-PCR data confirmed strong induction of TGF-β (SMAD6, JUN, SKIL) and endocytosis pathway (CXCR4, HSPA8, ADRB1) genes while downregulation of cell cycle genes (CDC27 and CDC23). Interestingly, use of TGF-β inhibitor, SB-431542, increased CHIKV mediated cell death. Overall, this study aims at providing the first complete transcriptome signature of host response upon CHIKV infection to aid identification of possible biomarkers and therapeutic targets.
Chikungunya-specific IgG and neutralizing antibody responses in natural infection of Chikungunya virus in children from India
Chikungunya virus (CHIKV) infection is endemic in many different countries. CHIKV outbreaks are emerging in new areas and re-emerging in previously exposed geographical regions, thus making it a significant public health concern. CHIKV infections are often clinically inapparent, especially in children, which poses a challenge to testing and evaluating any vaccine. During CHIKV infection, CHIKV-specific antibodies are produced, and some of these antibodies can neutralize viruses released from infected cells before they can enter uninfected cells. In this study, we evaluated IgG binding and neutralizing antibody responses in paired serum samples from CHIKV-infected children and those with other febrile illness, using a recombinant truncated E2 protein and whole CHIKV particles as test antigens. Antibody detection using the truncated E2 protein showed a significant overlap between CHIKV-infected subjects and those with other febrile illnesses. This overlap was greater when binding antibody titers were determined using fixed CHIKV particles as the test antigen. Acute- and convalescent-phase sera collected from children after CHIKV infection showed significant differences in their neutralizing capacity. The neutralizing and binding antibody response showed a significant positive correlation. We detected IgG antibodies in most cases during the acute phase of infection. This was observed at two different geographical locations, one of which is not considered highly endemic. Conventional wisdom would suggest this to be a marker of re-infection (secondary infection). However, dissenting opinions have been voiced in other viral diseases (such as Ebola) where studies have detected IgG in acute illness. In the absence of any significant body of work documenting secondary CHIKV infections, we believe further work is needed to understand the early IgG response that we observed.
Shared germline-biased plasmablast clonotypes shape early neutralizing antibody responses during acute Chikungunya virus infection
Chikungunya virus (CHIKV) infection causes acute febrile illness and severe joint inflammation, with some patients progressing to chronic arthritis. Early isotype-switched neutralizing antibody (nAb) responses have been associated with protection from chronic arthritis, but the cellular and molecular features of these early responses remain poorly defined. We performed single-cell sorting of plasmablasts (PBs) from patients with acute CHIKV infection and generated 94 human monoclonal antibodies (mAbs). These were evaluated for binding to CHIKV-infected Vero cells and to purified CHIKV, neutralizing activity, isotype distribution, somatic hypermutation (SHM), and immunoglobulin gene usage. PB-derived antibodies were compared with CHIKV-specific memory B cell (MBC)-derived antibodies from seropositive individuals. Over one-third of PB-derived mAbs recognized CHIKV-infected Vero cells, and most were class-switched. Among these, 12 bound to purified CHIKV, and 6 of them showed neutralizing activity. All PB-derived nAbs had germline-like sequences with little to no SHM, and four displayed shared clonotypic features, including a conserved `WEL' motif in the CDRH3 region. Conversely, CHIKV-specific MBC-derived nAbs showed diverse but often higher levels of SHM, consistent with affinity maturation. Together, our findings suggest that early nAb responses generated during acute CHIKV infection are germline-biased with overlapping features suggestive of convergent clonotype responses, providing a cellular and molecular basis of the early protective humoral immunity.
Diversity of rotavirus genotypes circulating in children < 5 years of age hospitalized for acute gastroenteritis in India from 2005 to 2016: analysis of temporal and regional genotype variation
Background From 2016, the Government of India introduced the oral rotavirus vaccine into the national immunization schedule. Currently, two indigenously developed vaccines (ROTAVAC, Bharat Biotech; ROTASIIL, Serum Institute of India) are included in the Indian immunization program. We report the rotavirus disease burden and the diversity of rotavirus genotypes from 2005 to 2016 in a multi-centric surveillance study before the introduction of vaccines. Methods A total of 29,561 stool samples collected from 2005 to 2016 (7 sites during 2005–2009, 3 sites from 2009 to 2012, and 28 sites during 2012–2016) were included in the analysis. Stools were tested for rotavirus antigen using enzyme immunoassay (EIA). Genotyping was performed on 65.8% of the EIA positive samples using reverse transcription- polymerase chain reaction (RT-PCR) to identify the G (VP7) and P (VP4) types. Multinomial logistic regression was used to quantify the odds of detecting genotypes across the surveillance period and in particular age groups. Results Of the 29,561 samples tested, 10,959 (37.1%) were positive for rotavirus. There was a peak in rotavirus positivity during December to February across all sites. Of the 7215 genotyped samples, G1P[8] (38.7%) was the most common, followed by G2P[4] (12.3%), G9P[4] (5.8%), G12P[6] (4.2%), G9P[8] (4%), and G12P[8] (2.4%). Globally, G9P[4] and G12P[6] are less common genotypes, although these genotypes have been reported from India and few other countries. There was a variation in the geographic and temporal distribution of genotypes, and the emergence or re-emergence of new genotypes such as G3P[8] was seen. Over the surveillance period, there was a decline in the proportion of G2P[4], and an increase in the proportion of G9P[4]. A higher proportion of mixed and partially typed/untyped samples was also seen more in the age group 0–11 months. Conclusions This 11 years surveillance highlights the high burden of severe rotavirus gastroenteritis in Indian children < 5 years of age before inclusion of rotavirus vaccines in the national programme. Regional variations in rotavirus epidemiology were seen, including the emergence of G3P[8] in the latter part of the surveillance. Having pre-introduction data is important to track changing epidemiology of rotaviruses, particularly following vaccine introduction.
Chikungunya Infection in India: Results of a Prospective Hospital Based Multi-Centric Study
Chikungunya (CHIKV) has recently seen a re-emergence in India with high morbidity. However, the epidemiology and disease burden remain largely undetermined. A prospective multi-centric study was conducted to evaluate clinical, epidemiological and virological features of chikugunya infection in patients with acute febrile illness from various geographical regions of India. A total of 540 patients with fever of up to 7 days duration were enrolled at Karnataka Institute of Medical Sciences (KIMS), Karnataka (South); Sawai Man Singh Medical College (SMS) Rajasthan (West), and All India Institute of Medical Sciences (AIIMS) New Delhi (North) from June 2008 to May 2009. Serum specimens were screened for chikungunya infection concurrently through RT-PCR and serology (IgM). Phylogenetic analysis was performed using Bioedit and Mega2 programs. Chikungunya infection was detected in 25.37% patients by RT-PCR and/or IgM-ELISA. Highest cases were detected in south (49.36%) followed by west (16.28%) and north (0.56%) India. A difference in proportion of positives by RT-PCR/ELISA with regard to duration of fever was observed (p<0.05). Rashes, joint pain/swelling, abdominal pain and vomiting was frequently observed among chikungunya confirmed cases (p<0.05). Adults were affected more than children. Anti-CHIK antibodies (IgM) were detected for more than 60 days of fever onset. Phylogenetic analysis based on E1 gene from KIMS patients (n = 15) revealed ∼99% homology clustering with Central/East African genotype. An amino acid change from lysine to glutamine at position 132 of E1 gene was frequently observed among strains infecting children. The study documented re-emergence of chikungunya in high frequencies and severe morbidity in south and west India but rare in north. The study emphasizes the need for continuous surveillance for disease burden using multiple diagnostic tests and also warrants the need for an appropriate molecular diagnostic for early detection of chikungunya virus.
First study conducted in Northern India that identifies group C rotavirus as the etiological agent of severe diarrhea in children in Delhi
Background Group C Rotavirus (RVC) is an enteric pathogen responsible for acute gastroenteritis in children and adults globally. At present there are no surveillance studies on group C Rotaviruses in India and therefore their prevalence in India remains unknown. The present study aimed to evaluate group C rotavirus infection among <5 years old children hospitalized with acute gastroenteritis in New Delhi. Methods A total of 350 fecal specimens were collected during September 2013 to November 2014 from <5 years old diarrheal patients admitted at KSCH hospital, Delhi. The samples found negative for group A rotavirus ( N  = 180) by Enzyme immunoassay were screened for group C rotavirus by RT-PCR with VP6, VP7 and VP4 gene specific primers. The PCR products were further sequenced (VP6, VP7, VP4) and analyzed to ascertain their origin and G and P genotypes. Results Six out of 180 (group A rotavirus negative) samples were found positive for group C rotavirus by VP6 gene specific RT-PCR, of which 3 were also found positive for VP7 and VP4 genes. Phylogenetic analysis of VP7 and VP4 genes of these showed them to be G4 and P[2] genotypes. Overall, the nucleotide sequence data (VP6, VP7 and VP4) revealed a close relationship with the human group C rotavirus with no evidence of animal ancestry. Interestingly, the nucleotide sequence analysis of various genes also indicated differences in their origin. While the identity matrix of VP4 gene ( n  = 3) showed high amino acid sequence identity (97.60 to 98.20%) with Korean strain, the VP6 gene ( n  = 6) showed maximum identity with Nigerian strain (96.40 to 97.60%) and VP7 gene ( n  = 3) with Bangladeshi and USA strains. This is true for all analyzed samples. Conclusion Our study demonstrated the group C rotavirus as the cause of severe diarrhea in young children in Delhi and provides insights on the origin of group C rotavirus genes among the local strains indicating their source of transmission. Our study also highlights the need for a simple and reliable diagnostic test that can be utilized to determine the disease burden due to group C rotavirus in India.
Protein Sequence Retrieval and Phylogenetic Analysis of Various α-amylase Producing Bacillus species
Due to advancements made in biotechnology, genetic manipulation and medium optimization has been possible and that has led to high yield in enzyme especially α-amylase with improvised properties that find its application suitably in detergent, laundry and starch processing industries. Various species of the genus Bacillus produce α-amylases with varying properties. The amylases that find application miraculously in different fields can be easily obtained from plant, animal and microbial sources. Out of the three sources, microbial source especially the genus Bacillus is extensively studied for its ability to produce the enzyme. In the present study, protein sequences of primarily identified α-amylase producing Bacillus spp. were retrieved from protein database of National Center for Biotechnology Information. Multiple sequence alignment was performed and construction of Phylogenetic tree among these sequences was carried out using Neighbor-joining method; data sets based on 1000 re-samplings in molecular genetics evolutionary analysis.
The Secondary Structural Models (16S rRNA) of Alkaline Amylase Producing Bacillus species strain SP-RM2: Phylogenetics Analysis
Bacterial isolates especially the Bacillus strains have gained an edge over other organisms across the globe. Enzymes derived from alkaliphilic Bacillus make up about half of the total industrial enzyme market. Detergents, textiles, starch, baking and animal feed are the major industries which utmost produce and use enzymes. Alkaline α amylases are among the most important enzymes and are of great significance for biotechnology. In the present investigation, one alkalitolerant bacterial isolate coded RM2 was characterized by partial 16S rRNA gene sequencing due to its ability to produce alkaline amylase at pH 8.0. The amplified consensus 16S rRNA gene sequence (RM2) was further searched against nucleotide database using Blast program to find out its potential homologs. Twelve closest bacterial spp. were identified and the phylogenetic analysis revealed the target bacteria (RM2) isolate belong to genus Bacillus. RM2 has shown relatedness to Bacillus flexus strain IFO15715 in sequence based divergence study whereas secondary structure of 16S r-RNA of Bacillus sp. strain SP-RM2 did not show any relatedness to Bacillus flexus strain IFO15715 even though they shared sequence based similarity.