Sandeepan Mukherjee

Prevalence of leptospirosis among dogs and rodents and their possible role in human leptospirosis from Mumbai, India

A total of 100 blood and 18 urine samples of rodents and suspected dogs were collected from Mumbai, India during 2006-2008. In order to determine the role of animals in transmission of the disease to humans, all the samples were screened retrospectively by real-time polymerase chain reaction for leptospiral DNA and antibodies were detected using microscopic agglutination test. Leptopsiral DNA was detected from two blood and five urine samples from rodents. Of a total of 71 rodent and dog samples investigated for anti-Leptospira antibodies, 14 (19.7%) were positive. Pyrogenes was the predominant serovar found in 100.0% (7/7) and 85.7% (6/7) from suspected canine cases and rodents, respectively; followed by Icterohemorrhagiae, which was found in one rodent sample 14.28% (1/7). The study proves that there is high prevalence of leptospirosis in rodents and dogs in this region, which proves possible role of these animals in transmission of leptospires to humans. Hence it is imperative to necessary control measures to prevent human leptospirosis.

Evaluation of In vitro antiviral activity of Datura metel Linn. against rabies virus

Objective: The soxhlet and cold extracts of Datura metel Linn. were evaluated for in vitro antirabies activity. Materials and Methods: Soxhlet and cold extraction method were used to extract Datura (fruit and seed) extracts. In vitro cytotoxicity assay was performed by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide assay. Based on the CC50 range, the in vitro antirabies activity of the extracts was screened by rapid fluorescent focus inhibition test and molecular method. Results: The Datura (fruit and seed) extracts were not cytotoxic below 5 mg/ml (CC50). Titer of 10−4 rabies virus challenge virus standard (RV CVS) (1 50% tissue culture infective dose [1 TCID50]) was obtained by RFFT method and the challenge dose of 10 TCID50 was used for antirabies assay. Datura fruit and seed (soxhlet and cold) extracts showed 50% inhibition of RV CVS at 2.5 mg/ml and 1.25 mg/ml (inhibitory concentration 50% [IC50]), respectively. The tested extracts showed selectivity index (CC50/IC50) ranging from 2 to 4. The viral RNA was extracted and real-time reverse transcription-polymerase chain reaction was performed which also revealed a 2-fold reduction of viral load at 1.25 mg/ml of the Datura seed (soxhlet methanolic and cold aqueous) extracts. Conclusion: To the best of our knowledge, this is the first study of in vitro antiviral activity of D. metel Linn. against rabies virus. Datura seed extracts have a potential in vitro antirabies activity and, in future, can be further screened for in vivo activity against rabies virus in murine model.

Evaluation of Neuroimmunomodulatory Activity of Recombinant Human Interferon α

Objective: Recombinant human interferon (rhIFN)-α is a potent immunoregulator having a wide range of therapeutic applications. In the present study, rhIFN-α was evaluated for its neuroimmunomodulatory activity. Method: Dose-dependent gene expression of cytokines and chemokines in the brain of rhIFN-administered mice was studied using real-time SYBR green PCR. Results: Statistically significant increase in expression levels of tumor necrosis factor-α, interleukin (IL)-6, IL-1β and IFN-γ were observed. Conclusion: The findings indicate that rhIFN-α may be used at an optimized dose to cause appropriate neuromodulation of cytokine/chemokine secretion that can aid in the development of therapeutic approaches for many infectious diseases of the central nervous system for which therapies are lacking.

Prevalence of influenza virus among the paediatric population in Mumbai during 2007-2009

PURPOSE Influenza has a major impact on public heath, annually affecting 15-20% of the global population. Information on the activity of influenza virus in Mumbai is limited. The present study was carried out to determine the prevalence of influenza viruses causing acute respiratory infections in children by molecular methods. OBJECTIVE To study the prevalence of influenza viruses among the paediatric population in Mumbai by real-time reverse-transcriptase polymerase chain reaction (rRT-PCR). MATERIALS AND METHODS From July 2007 to July 2009, 100 respiratory samples (nasal and throat swabs) were collected from paediatric patients with acute respiratory symptoms. attending out patients department, and admitted to the paediatric wards of B. J. Wadia Hospital for Children, Mumbai. The samples were collected and processed as per World Health Organization (WHO) guidelines. Viral RNA was extracted and one-step rRT-PCR was performed to detect influenza type A (H1 and H3) and influenza type B virus. RESULTS Out of 100 samples processed by rRT-PCR, a total of 11 samples (11%) were positive for influenza virus. The typing for influenza A subtypes showed 1% (1) positivity for H1 and 5% (5) positivity for H3 subtypes and 5% (5) samples tested positive for influenza type B virus. CONCLUSION It was observed that both influenza type A and B viruses were prevalent in Mumbai during the study period. Such surveillance data are important in the early detection of any antigenic variants that may be helpful in global influenza vaccine preparation and for any pandemic preparedness activity.

Molecular characterization of nucleoprotein gene of rabies virus from Maharashtra, India

Context: Rabies poses a serious public health concern in developing countries such as India. Aims: The study focuses on molecular diagnosis of street rabies virus (RABV) from human clinical specimens received from Maharashtra, India. Materials and Methods: Nucleoprotein gene from eight (of total 20 suspected samples) rabies cases that tested positive for rabies antigen using reverse transcriptase-polymerase chain reaction (RT-PCR) were sequenced. Results: Sequence analysis using basic local alignment search tool (BLAST) and multiple sequence alignment (MSA) and phylogenetic analysis showed similarity to previously reported sequences from India and those of Arctic lineages. Conclusions: The circulating RABV strains in Maharashtra, India show genetic relatedness to RABV strains reported from Indo-Arctic lineages and India-South and Japan.

Media Optimization in Immobilized Culture to Enhance the Content of Curcumin in Curcuma longa (Zingiberaceae) and Protein Profile of Treated Samples in Static Culture

Plant cell culture system has served as an alternative to enhance the production of high value phyto-pharmaceuticals. In the described study, the immobilized culture and static culture of Curcuma longa (Zingiberaceae) were used to maximize the production of the bioactive compound Curcumin. It was observed that Curcumin content in vitro studies (static as well as immobilize cultures) were enhanced in one week old cultures fed with 5 mg/100 ml of media (Static 8.71%; immobilize 2.03%). Statistically significant (seven fold) enhancement in Curcumin content was obtained in one week old static culture, which was maintained on Zenk production media incorporated with cinnamic acid (Control 1.57% and induced 8.717%). Quantitative estimation was done using HPTLC analysis with standard Curcumin. To evaluate the effect of treatment on total protein in Curcumin biosynthesis, we have examined Curcumin content as well as the protein profile of treated samples of Curcuma longa static culture. All the treated samples were analysed using SDS-PAGE for their proteomic profiles. A 23,420 Da protein was prominently expressed in all samples which may be a glycine rich protein (works on defence mechanism). Treated samples exhibited decreased expression of the protein as compared to control. This may be attributed to the formation of Reactive Oxygen species (ROS) in culture condition due to high concentration of sucrose (5%) in the Curcuma longa culture media that is known to induce oxidative stress and subsequent increase in Curcumin production. Further investigation is required to understand the actual protein involvement in Curcumin synthesis in different cultures treated with different compounds. The study signifies the use of plant explants to develop immobilized and static cultures rather than callus in Zenk media, which reduce the time as well as expenditure. The proteomic profile of C. longa has been discussed earlier, but its effect on proteomic profile of in vitro treated samples is a new study.

Scorpion venom research around the world: Indian Red Scorpion

The Indian red scorpion is the medically most important scorpion on the Indian subcontinent, and a sting is often lethal without treatment, especially to young children. The correct identification of this species is Hottentotta tamulus; the more commonly known names, Buthus tamulus and Mesobuthus tamulus, and the suggestions of subspecies (Pocock) are incorrect and should be discouraged. Mild cases of H. tamulus envenoming show vasoconstriction and hypertension resulting from a massive release of catecholamines. Severe cases result in hypotension, pulmonary edema and myocardial dysfunction. Animal studies indicate that catecholamine release is as a consequence of prolonged sodium channel activation and potassium channel inhibition. Pulmonary edema results from many subcellular events, including kinin activation and stimulation of central serotonin receptor subtypes. Treatment with a specific antibody (with or without the a-adrenoreceptor blocker, prazosin, dependent on symptoms and clinical severity) is presently the preferred therapy. The few venom constituents that have been characterized in detail include iberiotoxin, a specific blocker of high conductance, calcium-activated potassium channels; tamapin, a specific blocker of low conductance calcium activated potassium channels (KCa 2.2 subtype); and the short peptide insectotoxins, ButaIT and BtlTx3.

Characterization of influenza virus among influenza like illness cases in Mumbai, India

The present study was carried out to monitor influenza viruses by identifying the virus and studying the seasonal variation during 2007–2009 in Mumbai. A total of 193 clinical respiratory samples (nasal and throat swab) were collected from patients having influenza like illness in Mumbai region. One-step real-time reverse-transcriptase PCR (rRTPCR) was used to detect Influenza type A (H1 and H3) and Influenza type B virus. Isolation of the virus was carried out using in vitro system which was further confirmed and typed by hemagglutination assay and hemagglutination inhibition assay. Out of 193 samples 24 (12.4 3%) samples tested positive for influenza virus, of which 13 (6.73 %) were influenza type A virus and 10 (5.18 %) were influenza type B virus, while 1 sample (0.51 %) was positive for both. By culture methods, 3 (1.55 %) viral isolates were obtained. All the three isolates were found to be Influenza type B/Malaysia (Victoria lineage) by Hemagglutination Inhibition Assay. The data generated from the present study reveals that both Influenza type A and B are prevalent in Mumbai with considerable activity. The peak activity was observed during monsoon season.

Assessment of In vivo Antiviral Potential of Datura metel Linn. Extracts against Rabies Virus

Objective: The soxhlet, cold, and ayurvedic extracts of Datura metel Linn. were evaluated for in vivo antirabies activity. Materials and Methods: Soxhlet and cold extraction method were used to extract Datura (fruit and seed) extracts, and ayurvedic extraction of Datura was prepared. In vivo toxicity assay was performed as per the OECD 420. LD50dose was calculated by Reed and Muench method. The in vivo antirabies activity was screened in Swiss albino mice with the virus challenge dose of 10 LD50 (intracerebrally) in both preexposure (PE) and postexposure treatment with oral administration of Datura extracts in Swiss albino mice and observed for 21 days. The virus load in the mice brain was evaluated by TCID50 titration method. Results: Datura (ayurvedic preparation) was found to be nontoxic up to 2000 mg/kg in Swiss albino mice, i.e., 60 mg/30 g of mice, when administered (0.5 ml) orally and observed till 21 days. Up to 20% survival rate on the test group (PE of Datura extracts) up to 14 days postinfection as compared to the virus control group (10 LD50) was observed. No survival rate was observed in the postexposure group of Datura extract; however, the survival time was increased by 4 days as compared to the virus control group. Viral load of the infected mice brain sample was estimated in vero cell line, and 3 log reduction in the virus titer was observed in text group as compared to the virus control, suggesting that Datura extract has an in vivo antirabies activity. Conclusion: To the best of our knowledge, this is the first study of in vivo antiviral activity of an ayurvedic preparation of D. metel Linn. against rabies virus. Datura extracts have a potential in vivo antirabies activity.

Expression of domain III of the envelope protein from GP-78: a Japanese encephalitis virus

Acute encephalitis caused by the Japanese encephalitis virus (JEV) represents a growing epidemic and is a cause for concern in Southeast Asia. JEV is transmitted to humans through the bite of the Culicine mosquito species. The virus genome comprising of an RNA strand also encodes the envelope protein (E) which surrounds the virus. The E protein aids in fusion of virus with the cellular membrane of the host cell with the help of three structurally distinct domains (DI, DII, DIII) that are connected by flexible hinge regions. Of these domains, DIII (JEV-DIII) has been reported to interact with the cellular membrane, aid viral entry and viral replication. Hence JEV-DIII has the potential to be an antigen that can provide immune protection to a JEV infection. In this study, we describe the cloning and expression of DIII of GP-78, a virulent strain of JEV prevalent in India. Our data clearly shows that JEV-DIII expressed from pVAC1 in HEK293T cells is membrane targeted. To our knowledge, this is the first demonstration of a recombinant construct that may block JEV entry into the cells and/or evoke specific antibodies against JEV. Future studies will reveal if our construct will elicit significant immune responses which will alleviate or ameliorate the pro-inflammatory responses induced by JEV.