Viremia and clinical manifestations in acute febrile patients of Chikungunya infection during the 2016 CHIKV outbreak in Delhi, India.

Background: Chikungunya virus (CHIKV) is an infectious agent that caused several outbreaks among different countries and affected approximately 1.3 million Indian populations. It is transmitted by Aedes mosquito-either A. albopictus or A. aegypti. Generally, the clinical manifestations of CHIKV infection involve high-grade fever, joint pain, skin rashes, headache, and myalgia. The present study aims to investigate the relationship between the CHIKV virus load and clinical symptoms of the CHIKV infection so that better patient management can be done in the background of the CHIKV outbreak as there is no licensed anti-viral drug and approved vaccines available against CHIKV. Methods: CHIKV RTPCR positive samples (n = 18) (Acute febrile patients having D.O.F ≤ 7 days) were taken for the quantification of CHIKV viremia by Real-Time PCR. Clinical features of the febrile patients were recorded during the collection of blood samples. Results: The log mean virus load of 18 RT-PCR-positive samples was 1.3 × 106 copies/mL (1.21 × 103-2.33 × 108 copies/mL). Among the observed clinical features, the log mean virus load (CHIKV) of the patients without skin rash is higher than in the patients with skin rash (6.61 vs 5.5, P = 0.0435). Conclusion: The conclusion of the study was that the patients with skin rashes had lower viral load and those without skin rashes had higher viral load.

Genotypic characterization of vancomycin-resistant Enterococcus causing urinary tract infection in northern India.

Background & objectives: Infections caused by vancomycin-resistant Enterococci are difficult to treat given the limited therapeutic alternatives. Different gene clusters are known to confer vancomycin resistance. vanA and vanB genes are transferable and are clinically relevant. This cross-sectional study aimed to identify the vancomycin-resistant genotypes in the strains causing urinary tract infection and also to test the in vitro efficacy of linezolid and pristinamycin against the vancomycin-resistant isolates. Methods: Antimicrobial resistance profile of 118 enterococcal isolates was evaluated. Minimum inhibitory concentration of vancomycin, teicoplanin and high-level gentamicin (HLG) was determined by micro broth dilution. The vancomycin-resistant isolates were tested against linezolid and pristinamycin by micro-broth dilution and E strip method. The presence of vancomycin-resistant genes was detected by multiplex polymerase chain reaction and was sequenced and analyzed. Results: Most commonly isolated species were Enterococcus faecalis (76.9%) and Enterococcus faecium (16.9%). It was found that 43 per cent of the isolates were resistant to HLG and 16.9 per cent to vancomycin. Higher resistance was seen against fluoroquinolones, erythromycin, tetracycline and ß-lactam drugs. However, 5.08 per cent strains were resistant to tigecycline. All vancomycin-resistant strains were sensitive to pristinamycin and one was resistant to linezolid. vanA and vanB gene were found in 15 and five isolates, respectively. The gene sequences were submitted to NCBI gene bank and accession numbers were obtained. Interpretation & conclusions: The present study showed prevalence of vanA and vanB genes carrying Enterococcus in a tertiary care centre in north India. The emergence of resistance against drugs such as tigecycline and linezolid is a topic of concern as it will be a therapeutic challenge for physicians.

High-level gentamicin resistance mediated by Aac(6')-Ie-aph(2")-Ia gene in Enterococcus species isolated from clinical samples in Northern India.

CONTEXT: Enterococci are known to cause life-threatening infections which are difficult to treat as the organism harbors innate resistance to many antibiotics and can amass resistance toward many others through plasmid-mediated genetic exchange. AIMS: The study evaluates the drug susceptibility profile of various Enterococcus species isolated from various patient specimens submitted for bacteriological analysis and check the incidence of aac(6') Ie-aph(2") Ia gene. SETTING AND DESIGN: This in vitro cross-sectional study was executed at bacteriology laboratory of a 470 bedded hospital in New Delhi. MATERIALS AND METHODS: Drug susceptibility testing was carried out on enterococcal isolates. High-level gentamicin-resistant (HLGR) isolates detected by micro broth dilution assay were then subjected to molecular detection of aac(6') Ie-aph(2") Ia gene. STATISTICAL ANALYSIS USED: The level of significance was established by Chi-square test. RESULTS: Among the 182 enterococcal stains detected, 76.9% were Enterococcus faecalis and 20.3% were Enterococcus faecium. 12.08% strains were vancomycin resistant. 39% expressed resistance toward high-level gentamicin (HLG) and this finding was significantly higher in E. faecium than E. faecalis. HLGR strains expressed a higher degree of resistance to other drugs in contrast to non-HLGR isolates. In 67 out of 71 HLGR isolates the bifunctional gene was detected. CONCLUSION: Considerable presence of HLG and vancomycin resistance in the clinical isolates is alarming and should be taken seriously. The study shows high dissemination of aac(6')-Ie-aph(2")-Ia gene among Enterococci isolated from the region.

Comparative kinetic analysis of ascorbate (Vitamin-C) recycling dehydroascorbate reductases from plants and humans.

Ascorbate is an important cellular antioxidant that gets readily oxidized to dehydroascorbate (DHA). Recycling of DHA is therefore paramount in the maintenance of cellular homeostasis and preventing oxidative stress. Dehydroascorbate reductases (DHARs), in conjunction with glutathione (GSH), carry out this vital process in eukaryotes, among which plant DHARs have garnered considerable attention. A detailed kinetic analysis of plant DHARs relative to their human counterparts is, however, lacking. Chloride intracellular channels (HsCLICs) are close homologs of plant DHARs, recently demonstrated to share their enzymatic activity. This study reports the highest turnover rate for a plant DHAR from stress adapted Pennisetum glaucum (PgDHAR). In comparison, HsCLICs 1, 3, and 4 reduced DHA at a significantly lower rate. We further show that the catalytic cysteine from both homologs was susceptible to varying degrees of oxidation, validated by crystal structures and mass-spectrometry. Our findings may have broader implications on crop improvement using pearl millet DHAR vis-à-vis discovery of cancer therapeutics targeting Vitamin-C recycling capability of human CLICs.

Phylogenetic analysis of VP7 and VP4 genes of the most predominant human group A rotavirus G12 identified in children with acute gastroenteritis in Himachal Pradesh, India during 2013-2016.

G12 strains are now considered to be the sixth most prevalent human rotaviruses globally. India has introduced rotavirus vaccine Rotavac® into the national immunization program in 2016 and Himachal Pradesh (HP) is the first state to launch it. During epidemiological rotavirus surveillance in HP, predominance of G12 rotaviruses was observed. This study investigated the genetic variability and evolution of HP G12 strains (n = 15) associated with P-genotypes P[6], P[4], and P[8] identified between 2013 and 2016. Phylogenetic analysis of VP7 gene revealed that all characterized G12 strains clustered in lineage-III and diversified into three subclusters indicating that these strains may have originated from three different ancestral G12 strains. The comparative sequence analysis of HP strains with Rotavac® and Rotarix® vaccine strains revealed various amino acid substitutions in epitope regions of VP7 and VP4 proteins especially at the antibody neutralization sites. Only 12/29 VP7 epitope residues and 2/25 VP4 epitope residues were found to be conserved between HP rotavirus strains and vaccine strains. Both long and short electropherotypes were observed in G12P[4] strains, while a single long electropherotype was observed in G12P[6] strains. Children of ≤11 months were significantly infected with G12 rotaviruses. The frequency of vomiting episodes (≥5/day) was significantly higher in children infected with G12 rotavirus strains as compared to non-G12 rotaviruses (p = 0.0405). Our study provides the comprehensive data on clinical characteristics and evolutionary pattern of the G12 rotavirus, the most prevalent strain in HP and emphasizes the need to monitor these strains for inclusion in future vaccine.

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.

Genetic diversity of G9 rotavirus strains circulating among diarrheic children in North India: A comparison with 116E rotavirus vaccine strain.

The parental rotavirus strain 116E (G9P[11]) used to generate Rotavac® vaccine was isolated in 1986 in New Delhi. Thenceforward, there is no comprehensive report on diversity of G9 rotavirus strains from 116E; therefore, the present study evaluates the VP7 gene sequence diversity of G9 strains (retrieved from GenBank) from different geographical regions (1987-2016). Additionally, 22 recently collected G9 strains from Himachal Pradesh and Delhi (2013-2016) were included in the phylogenetic analysis. Interestingly, unlike 116E which belong to lineage-II all other G9 rotavirus including these 22 samples clustered together in a separate lineage (III). Further, six amino acid substitutions including one novel, K143M (epitope 7-2) different from 116E were detected mostly in the neutralization epitopes of VP7 protein (neutralization escape mutants). Overall, the accumulation of identified substitutions in VP7 epitopes and evolution of G9 strains in India may have impact on Rotavac® efficacy.

Epidemiology and molecular characterization of chikungunya virus from human cases in North India, 2016.

Chikungunya virus (CHIKV), an arthropod-borne Alphavirus is responsible for chikungunya disease. Arthralgia and arthritis are the major symptom. Some patients recover early while others for a very long time. This study provides, epidemiology and molecular characterization of three whole-genome sequences of CHIKV and assessed phylogenetic analysis, physiological properties, antigenicity, and B-cell epitope prediction by in silico. We report the clinical epidemiology of 325 suspected patients. Of these, 118 (36.30%) were confirmed CHIKV positive by either PCR or ELISA. Clinical analysis showed joint pain, joint swelling and headache were frequent and significant features. Phylogenie analysis showed the currently circulating strain is in close clustring to Africa, Uganda, and Singapore CHIKV strains. Molecular characterization by WGS was done. Thirty eight amino acid changes in the nonstructural proteins were found with respect to the S27 (ECSA) strain. Of these five located in nsP2. Similarly, 34 amino acid changes in structural proteins were observed. The major change was notice; in E3 protein hydropathicity -0.281 to -0.362, in E2 isoelectric point (pI) 8.24 to 8.37, instability index 66.08 to 71.062, aliphatic index varied from 74.69 to 68.59 and E3 75.79 to 70.05. In nsP1 protein pI varies from 6.62 to 8.04, while no other change was observed in structural and nonstructural protein. The linear B-cell epitopes, position, and number varied with the mutation. The molecular characterizations of WGS demonstrate the observation of protein, antigenicity with respect to the mutation.

Discovery of small molecule inhibitors of chikungunya virus proteins (nsP2 and E1) using in silico approaches.

Chikungunya virus (CHIKV) has emerged as a major viral threat, affecting over a million people worldwide per year. It is a vector borne disease transmitted to the human by Ades mosquitoes and primarily affect people by causing viral fever, severe joint pain and other symptoms, like rash, joint swelling, muscle pain and in rare cases can be fatal. CHIKV is a deadly virus, with its mutation rate found to be significantly higher as compared to other viruses. To date, there has been no reported FDA approved drug against this virus. Thus, keeping in mind the urgent need to scrutinize potential therapies against CHIKV, the present study identified twenty plant bioactive compounds that are available at low price and do not have associated adverse effect. For identification of active potentials molecules the pharmacoinformatics-based perspective was applied against CHIKV structural (E1) and non-structural (nsP2) proteins using molecular docking and scoring. The selected compounds were further studied for pharmacokinetics (PK) and pharmacodynamics (PD) associated parameters such as initial absorption, then distribution and later on metabolism excretion and toxicity (ADMET) profiles based on in silico study. The results reveal five potential lead compounds having high binding energy that can help in the development of commercial drugs with favorable ADMET characteristic.Communicated by Ramaswamy H. Sarma.

Epidemiology of rotavirus diarrhea among children less than 5 years hospitalized with acute gastroenteritis prior to rotavirus vaccine introduction in India.

BACKGROUND: Rotavirus is an important cause of severe diarrhea requiring hospitalization, accounting for approximately 78,000 deaths annually in Indian children below 5 years of age. We present epidemiological data on severe rotavirus disease collected during hospital-based surveillance in India before the introduction of the oral rotavirus vaccine into the national immunization schedule. METHODS: The National Rotavirus Surveillance Network was created involving 28 hospital sites and 11 laboratories across the four geographical regions of India. From September 2012 to August 2016 children less than 5 years of age hospitalized for diarrhea for at least 6 h, were enrolled. After recording clinical details, a stool sample was collected from each enrolled child, which was tested for rotavirus antigen using enzyme immunoassay (EIA). Nearly 2/3rd of EIA positive samples were genotyped using reverse transcription polymerase chain reaction to identify the G and P types. RESULTS: Of the 21,421 children enrolled during the 4 years surveillance, 36.3% were positive for rotavirus. The eastern region had the highest proportion of rotavirus associated diarrhea (39.8%), while the southern region had the lowest (33.8%). Rotavirus detection rates were the highest in children aged 6-23 months (41.8%), and 24.7% in children aged < 6 months. Although rotavirus associated diarrhea was seen throughout the year, the highest positivity was documented between December and February across all the regions. The most common rotavirus genotype was G1P[8] (52.9%), followed by G9P4 (8.7%) and G2P4 (8.4%). CONCLUSIONS: There is high burden of rotavirus gastroenteritis among Indian children below 5 years of age hospitalized for acute diarrhea thereby highlighting the need for introduction of rotavirus vaccine into the national immunization program and also for monitoring circulating genotypes.

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.

Biofilm synthesis and other virulence factors in multidrug-resistant uropathogenic enterococci isolated in Northern India.

Purpose: Enterococci express high degree of resistance towards wide range of antibiotics. Production of biofilm and many virulence factors along with drug resistance makes it difficult to eradicate the infection from urinary tract. The present study detected the expression of such factors including biofilm production by multidrug-resistant (MDR) enterococci. Materials and Methods: Drug susceptibility of 103 uropathogenic enterococci was performed followed by estimation of minimum inhibitory concentration of high-level gentamicin and vancomycin by microbroth dilution method. Vancomycin-resistant genes were detected by multiplex polymerase chain reaction. Production of virulence factors such as haemagglutination, caseinase, lipase, gelatinase, haemolysin and ß-lactamase was detected by phenotypic methods in MDR strains. Biofilm production was detected by calcofluor-white fluorescence staining and semi-quantitative adherence assay. Results: 45% and 18.4% of the isolates were high-level gentamicin-resistant and vancomycin-resistant enterococci (VRE), respectively. vanA gene was detected in 14 and vanB gene in 5 strains. Biofilm, caseinase and gelatinase were the most expressed virulence factor. Expression of caseinase, gelatinase and lipase was significantly higher in Enterococcus faecalis (P < 0.05). Expression of haemagglutination, gelatinase and haemolysin among the vancomycin-resistant isolates was significantly higher (P < 0.05). Conclusion: VanA and vanB are the prevalent genotypes responsible for vancomycin resistance. The high prevalence of MDR enterococcal strains producing biofilm and virulence determinants raises concern. asa1, hyl, esp, gelE, cyl and other genes are known to express these factors and contribute to biofilm formation. Most uropathogenic enterococci expressed biofilm at moderate level and can be detected effectively by calcofluor-white staining. No correlation was noted between vancomycin resistance and biofilm production.

Antibody response patterns in chikungunya febrile phase predict protection versus progression to chronic arthritis.

Chikungunya virus (CHIKV) infection causes acute febrile illness in humans, and some of these individuals develop a debilitating chronic arthritis that can persist for months to years for reasons that remain poorly understood. In this study from India, we characterized antibody response patterns in febrile chikungunya patients and further assessed the association of these initial febrile-phase antibody response patterns with protection versus progression to developing chronic arthritis. We found 5 distinct patterns of the antibody responses in the febrile phase: no CHIKV binding or neutralizing (NT) antibodies but PCR positive, IgM alone with no NT activity, IgM alone with NT activity, IgM and IgG without NT activity, and IgM and IgG with NT activity. A 20-month follow-up showed that appearance of NT activity regardless of antibody isotype or appearance of IgG regardless of NT activity during the initial febrile phase was associated with a robust protection against developing chronic arthritis in the future. These findings, while providing potentially novel insights on correlates of protective immunity against chikungunya-induced chronic arthritis, suggest that qualitative differences in the antibody response patterns that have evolved during the febrile phase can serve as biomarkers that allow prediction of protection or progression to chronic arthritis in the future.

Exploring rotavirus proteome to identify potential B- and T-cell epitope using computational immunoinformatics.

Rotavirus is the most common cause of acute gastroenteritis in infants and children worldwide. The functional correlation of B- and T-cells to long-lasting immunity against rotavirus infection in the literature is limited. In this work, a series of computational immuno-informatics approaches were applied and identified 28 linear B-cells, 26 conformational B-cell, 44 TC cell and 40 TH cell binding epitopes for structural and non-structural proteins of rotavirus. Further selection of putative B and T cell epitopes in the multi-epitope vaccine construct was carried out based on immunogenicity, conservancy, allergenicity and the helical content of predicted epitopes. An in-silico vaccine constructs was developed using an N-terminal adjuvant (RGD motif) followed by TC and TH cell epitopes and B-cell epitope with an appropriate linker. Multi-threading models of multi-epitope vaccine construct with B- and T-cell epitopes were generated and molecular dynamics simulation was performed to determine the stability of designed vaccine. Codon optimized multi-epitope vaccine antigens was expressed and affinity purified using the E. coli expression system. Further the T cell epitope presentation assay using the recombinant multi-epitope constructs and the T cell epitope predicted and identified in this study have not been investigated. Multi-epitope vaccine construct encompassing predicted B- and T-cell epitopes may help to generate long-term immune responses against rotavirus. The computational findings reported in this study may provide information in developing epitope-based vaccine and diagnostic assay for rotavirus-led diarrhea in children's.

Epidemiology and clinical features of rotavirus, adenovirus, and astrovirus infections and coinfections in children with acute gastroenteritis prior to rotavirus vaccine introduction in Meerut, North India.

There are limited reports on the etiology of multiple enteric viruses causing acute gastroenteritis (AGE) in North India. In the present study we have determined the prevalence of three enteric viruses, namely rotavirus, astrovirus (AstV) and adenovirus (AdV) in a total of 312 diarrheic children (<5 years) hospitalized at Lala Lajpat Rai Memorial Medical College, Meerut, Uttar Pradesh from August 2014 to July 2016; and results were compared with data from Delhi. The fecal samples were individually screened for group A rotavirus (RVA), AdV, and AstV using enzyme immunoassay kits. At least one viral agent was detected in 29.2% of 312 fecal specimens. RNA of rotavirus antigen-positive samples was extracted by TRIzol method. Rotavirus G/P genotyping was performed using seminested multiplex reverse transcriptase-polymerase chain reaction. RVA was the most predominant virus (18.3%) followed by AstV (12.5%), and AdV (9.9%). Coinfections were detected in 10.6% cases and the most common coinfection in diarrheic children was RVA combined with AstV (36.4%). Overall, the enteric viruses were found most prevalent in the 6 to 11 months age group (P = .01). Increased duration of vomiting (≥3 days) was significantly (P = .04) associated with AdV infection (61.3%) as compared with AstV (30.76%) and rotavirus (26.31%). G1P[8] was detected throughout as the most prevalent rotavirus strain (10.5%). Unusual RV strains like G2P[6] and G2P[8] were also detected. Of note G3, G4, and G12 rotavirus were detected for the first time in Meerut. This is the first report that demonstrated the important contribution of multiple enteric viruses causing AGE in young children in this part of Uttar Pradesh (Meerut).

Production, characterization and application of thermostable, alkaline α-amylase (AA11) from Bacillus cereus strain SP-CH11 isolated from Chilika Lake.

An alkaliphile bacterial strain designated as CH11 was isolated from the sediments of Chilika Lake, Odisha. The isolate showed stupendous growth and production of α-amylase at pH 10.0. Through 16S rRNA gene based molecular technique this isolate was identified as Bacillus cereus strain SP-CH11 having GenBank Accession No. KT992791. Homogenous ~55 kDa extracellular α-amylase was extracted with 241.304, 26.26 and 3.2-fold acceleration in specific activity, purification fold and yield respectively. The alkaline α-amylase AA11 was further characterized. At pH 9.0 the purified enzyme AA11 was highly stable while retaining 88-100% functional viability at temperature range from 35 to 65 °C, confirming its thermostability nature. It showed stability with powdered and liquid detergents at 7 mg/mL and 100-fold dilutions respectively. AA11 efficiently removed the starch stain from cotton fabrics. The findings of this study indicate that the isolate CH11 is a source of novel alkaline α-amylase that has promising application in food and detergent industries.

Exploration of detergent-stable alkaline α-amylase AA7 from Bacillus sp strain SP-CH7 isolated from Chilika Lake.

An alkali-tolerant bacterial isolate CH7 was isolated from Chilika Lake sediments that produced α-amylase at high pH. It showed >15 mm zone diameter at pH 11.0 on agar plate containing 1% starch hence considered for study. It survived up to 10% NaCl concentration on nutrient agar plates. Taxonomically it was identified as Bacillus sp. strain SP-CH7 having GenBank Accession No. KU761266. Optimized alkaline α-amylase, AA7 showed 202.857 specific activity (IU/mg), 65.95 purification fold and 25.94 yield (%) respectively. Molecular weight was revealed by SDS-PAGE as 55 kDa. The Km value was 0.688 mg/mL and so was 2.342 IU/mL Vmax obtained for AA7 towards starch. This enzyme showed 92.79% and 91.14% stability at pH 11.0 and 13.0 respectively and up to 75 °C stability was also noted. Enzyme activity was stimulated by Co2+, Na+ and Ca2+. It was stable in presence of EDTA, SDS, all the powdered detergents as well as with liquid detergent "Ezee" which vouches for its potential application in Detergent industries.

Epidemiology and genetic diversity of group A rotavirus in acute diarrhea patients in pre-vaccination era in Himachal Pradesh, India.

Acute gastroenteritis due to Group A rotaviruses remains the leading cause of mortality and morbidity in children in developing countries. India introduced its indigenous rotavirus vaccine Rotavac® in 2016 and Himachal Pradesh (HP) the first state to launch it. The present study aimed to evaluate rotavirus strain diversity associated with AGE prior to vaccine introduction in HP. A total of 331 fecal specimens collected from diarrheic children hospitalized at RPGMC Tanda, HP between July-2014 and June-2016 were screened for RVA by EIA. Rotavirus RNA was extracted by TRIZOL method and analyzed by RNA-PAGE. G/P typing was performed using semi-nested multiplex reverse transcriptase PCR. Rotavirus was detected in 45% (n = 149/331) of diarrheic children, with highest rate observed in the 6-11 months age group (47%). Vomiting was found more frequently associated with RV-infection. Among G-types, G12 was found most prevalent (33.1%) followed by G1 (28.4%), G9 (12.2%), G2 (9.5%), G3 (3.4%) and G10 (2.7%). G4 (0.7%) strains were rarely detected. Among P-types, P[6] was the most prevalent (40.5%) followed by P[8] (29.1%) and P[4] (14.2%). Of note, genotypes G3 and P[11] were detected for the first time in HP. Among G/P combinations, G12P[6] was most prevalent (30.4%) followed by G1P[8] (20.3%), G2P[4] (4.7%), G1P[6] (3.4%) and G3P[8] (2.7%). Interestingly, our study observed high percentage of unusual strains (14.2%) namely G9P[4], G2P[6], G2P[8], G12P[4] and G1P[11]. The regionally common strains G3P[6], G4P[6], G9P[6], G9P[8], G10P[6], G10P[8] and G12P[8] strains were very rarely detected. Of interest, RNA migration pattern of G1P[8] was DS-1 like and genomic heterogeneity was observed within G12P[4] strains with both long and short electropherotypes. Our study highlights rich genetic diversity with emergence of rare rotavirus strains circulating in HP and provides baseline data prior to Rotavac® introduction that will help to gauge the impact of the Rotavac® vaccine in HP.

Evaluation of medicinal herbs for Anti-CHIKV activity.

Chikungunya, a mosquito-borne viral disease is now a global public health problem. In tropical countries such as India, periodic chikungunya outbreaks can occur due the high prevalence of the mosquito vector, circulation of virus and the high population density. To curtail the virus in outbreak situation, a ready to use drug for chikungunya is necessary. Using the literature mentioned plant extracts, we used four assays to screen and identify indigenous plants with CHIKV inhibitory activity. Our results showed that the aqueous extract of five plant extracts exhibited anti-CHIKV activity by inhibiting viral attachment, four plant extracts exhibited replication inhibition through inhibition of helicase activity, two plants showed inhibition of protease activity. Two plant extracts showed both viral attachment inhibition and replication inhibition and also exhibited dose dependent response in virus replication inhibition assay. These findings warrant further investigation to standardize these plant extracts as antiviral formulation for chikungunya infection.

Virus load and clinical features during the acute phase of Chikungunya infection in children.

BACKGROUND: 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. OBJECTIVES: 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. STUDY DESIGN: 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. RESULTS: 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). CONCLUSION: Patients with myalgia had lower virus load and those without myalgia had a higher virus load.