International Journal of Research and Development in Pharmacy & Life Sciences, Oct 1, 2018
Dengue virus is mosquito-borne virus that manifests itself in human infections with dengue fever ... more Dengue virus is mosquito-borne virus that manifests itself in human infections with dengue fever (DF) to dengue hemorrhagic fever (DHF). DHF can lead to development of dengue shock syndrome (DSS). RNAi works by silencing the target gene expression using siRNA. Hence there arises an urgent need to design potential siRNA against the target sequence of dengue virus gene to control its replication and pathogenicity. This study is aimed to predict self-potential designed siRNA-based therapeutics that might be used for the treatment of dengue virus infection. The prediction of potential siRNA was done by using various computational tools as searching target sequences, multiple sequences alignment, secondary structure prediction, siRNA-target sequence interaction prediction and finally the evaluation of effectiveness of predicted siRNA. Ten pair of potential siRNAs were predicted and designed rationally for silencing five target genes (Capsid, CprM, NS1, NS3 and NS5) of dengue virus used in the study through RNAi technology. The outcomes of this study are ten pair of potential siRNA molecules which might be used as a potential antiviral RNA based therapeutics to suppress the dengue virus replication.
International journal of innovation and scientific research, 2015
Chronic obstructive pulmonary disease is the leading cause of fatality. The course of COPD is fol... more Chronic obstructive pulmonary disease is the leading cause of fatality. The course of COPD is followed by episodes of acute deterioration in respiratory health, referred as 'exacerbations'. Acute exacerbations of COPD contribute substantially to the morbidity and mortality due to number of infectious agents including bacteria, viruses, or both. Therefore, we planned a case control study to know the association of respiratory viruses especially HRSV genotype with acute exacerbation of COPD, if any. This is a prospective case-control study with two groups of patients (AECOPD and stable COPD). Nasopharyngeal aspirate were tested for the detection of Human Respiratory Syncytial Virus; Influenza Viruses; Human Metapneumovirus; Adeno Virus; Human Boca Virus and Parainfluenza Virus 1,2,3,4 by real time PCR. Respiratory viruses are more often found in case group (AECOPD patients) 45/ 234 patients (19.23%) than in control group (stable COPD), 8/100 patients (8%; P=0.0330). In case gr...
The study was conducted to analyze changes in HA and NA genes of pandemic pH1N1 strains, collecte... more The study was conducted to analyze changes in HA and NA genes of pandemic pH1N1 strains, collected from North Indian tertiary care hospital during 2015 and studied the nucleotide change since 2009. Differences in clinical features of cases positive and negative for influenza viruses were also studied. All samples referred for H1N1 testing, were tested for influenza viruses. Randomly selected 25 strains of pH1N1 were studied for nucleotide sequences of HA & NA gene. The nucleotide changes occurring since 2009 were studied by mutational and phylogenetic analysis. Clinical details of cases were recorded and analysed. A total of 3319 cases of acute respiratory infections (ILI/SARI) were tested for influenza viruses during Jan to April 2015, of which 815 cases tested positive for pH1N1. Nucleotide variation of 2015 strains, from influenza A/California/07/2009 strain at HA1 and NA1 gene was 1.9% and 3.8% respectively. Both HA1 and NA1 coding sequence showed eight mutations. Four of HA1(K1...
Genomic variability makes Influenza A virus (IAV) 'the least susceptible' to existing vaccines or... more Genomic variability makes Influenza A virus (IAV) 'the least susceptible' to existing vaccines or anti-influenza drugs. siRNA targeting viral gene silents the gene by cleaving mRNA. Present study aimed to develop siRNA targeting polymerase basic 1 (PB1) gene and to validate its efficiency in vitro. siRNA was designed rationally, targeting the most conserved region of PB1 gene of IAV strains. Total 147 strains worldwide and 42 Indian strains, when aligned, showed seven sets of conserved regions (> 30 bp stretch and < 5% mismatches). To choose the most efficient siRNA, three levels screening method was developed. Finally one pair of siRNA was chosen due to its unique position in conserved region. siRNA efficacy was confirmed in vitro on Madin Darby Canine Kidney (MDCK) cell line propagating two clinical isolates i.e. Influenza A/H3N2 [A/India/LKO864/ 2011(H3N2)] and Influenza A/pandemicH1N1 [A/India/LKO2151/2012(H1N1)]. The longest ORF was targeted by the selected siRNA, which showed 57 % inhibition in replication of Influenza A/pdmH1N1 and 60.6 % inhibition in replication of Influenza A/H3N2 at 72 hpi and 48 hpi respectively on MDCK cell line. This study shows that siRNA targeting PB1 may be moderately effective in controlling IAV replication so can be used as anti-IAV therapeutic agent.
Multiplex RT-PCR assays are widely used tools for detection of hepatitis viruses, but none of the... more Multiplex RT-PCR assays are widely used tools for detection of hepatitis viruses, but none of them provide quality check of sample. In the present study we developed a single-step triplex real-time polymerase chain reaction (PCR) assay for detection of Hepatitis B Virus (HBV) and Hepatitis C Virus (HCV) with sample quality check, by using β-actin as housekeeping gene. The primers and probes were self-designed and assay was standardized. Assay was also destined to quantitate copy numbers of HBV and HCV. This novel assay was sensitive, specific, and reproducible for detection of HBV and HCV in serum/plasma. The assay also detected all genotypes of HBV and HCV. The detection limit was 60IU/mL for HBV and 20IU/mL for HCV. This assay is the first assay developed on single-step platform for nucleic acid detection of HBV and HCV with an extra edge over all other assays by providing inbuilt check for quality of sample.
Hepatitis B virus (HBV) is among the most common causes of liver cirrhosis. We report the full-ge... more Hepatitis B virus (HBV) is among the most common causes of liver cirrhosis. We report the full-genome sequences of seven molecular clones of HBV genotype A, amplified from an HBV-infected North Indian patient. This is probably the first report of the HBV genome sequencing using Ion Torrent from India.
The genomic variability of Influenza A virus (IAV) makes it difficult for the existing vaccines o... more The genomic variability of Influenza A virus (IAV) makes it difficult for the existing vaccines or anti-influenza drugs to control. The siRNA targeting viral gene induces RNAi mechanism in the host and silent the gene by cleaving mRNA. In this study, we developed an universal siRNA and validated its efficiency in vitro. The siRNA was designed rationally, targeting the most conserved region (delineated with the help of multiple sequence alignment) of M gene of IAV strains. Three level screening method was adopted, and the most efficient one was selected on the basis of its unique position in the conserved region. The siRNA efficacy was confirmed in vitro with the Madin Darby Canine Kidney (MDCK) cell line for IAV propagation using two clinical isolates i.e., Influenza A/H3N2 and Influenza A/pdmH1N1. Of the total 168 strains worldwide and 33 strains from India, 97 bp long (position 137-233) conserved region was identified. The longest ORF of matrix gene was targeted by the selected si...
International Journal of Applied Sciences and Biotechnology, 2014
Introduction: In spite of the discovery of viral culture technology about a century ago, its appl... more Introduction: In spite of the discovery of viral culture technology about a century ago, its application in diagnostic labs is being used since 1970s. It served as the "gold standard" for virus detection for long. In recent years, rapid, technically less challenging, sensitive and highly specific viral identification is possible by molecular tools. Hence, the purpose of this study was to analyze the importance of real time PCR over virus culture in diagnosis of Influenza virus infections, the biggest viral challenge of present India, a developing country, so that prompt and correct diagnosis can help physicians as well as the policy makers to control the virus spread. Objective: To study the feasibility of real time PCR vis a vis viral culture technique and evaluate the utility of these methods for laboratory diagnosis of Influenza virus infections. Methodology: The study was conducted in Grade I Virology Diagnostic laboratory, Dept of Microbiology, KGMU, Lucknow. We used ...
During the post influenza pandemic period, continuous surveillance of influenza virus and its sub... more During the post influenza pandemic period, continuous surveillance of influenza virus and its subtypes is mandatory to help the policy makers to take effective and appropriate decisions. Therefore, this study was planned to determine the pattern of influenza virus activity in context to various meteorological and clinical parameters in and around Lucknow, Uttar Pradesh, India, during post pandemic period August 2010 - September 2012. Nasal swabs/throat swabs/nasopharyngeal aspirates of 2669 patients were collected. One-step real time PCR for detection of influenza virus was done according to the Centers for Disease Control and Prevention (CDC) protocol. Influenza positivity was 15.8 per cent (423/2669) in symptomatic patients. Of the 423 total positives, 192 (7.2%) were influenza A and 231 (8.7%) were influenza B. Positivity for influenza virus was significantly (P=0.001, OR=2.9, CI=1.9-4.3) higher in patients with Influenza like illness (ILI) (17.4%, 396/2271) than those with sever...
The human respiratory syncytial virus (HRSV) is a community-acquired virus that mainly causes acu... more The human respiratory syncytial virus (HRSV) is a community-acquired virus that mainly causes acute respiratory tract infection in infants and children. HRSV is increasingly recognized as an important nosocomial pathogen causing morbidity in immunocompromised patients. Here, we are reporting a hospital outbreak of HRSV during summer in children receiving chemotherapy for haematological malignancies. Prompt detection and timely preventive measures could abort the devastating outbreak. In the month of June 2013, seven children from paediatric oncology Ward at King George Medical University presented with respiratory signs and symptoms in span of 3 days. Nasal and throat swabs of children were tested for the presence of HRSV, human metapneumovirus and their subtypes A and B, influenza A and B, measles, parainfluenza virus (1, 2, 3 and 4), adenovirus, and human bocavirus by real-time polymerase chain reaction. A segment of F gene of HRSV was amplified and sequenced for phylogenetic anal...
Human parvovirus 4 (Parv4) is a relatively new virus. Association of this virus with any human di... more Human parvovirus 4 (Parv4) is a relatively new virus. Association of this virus with any human disease is yet to be established. We detected human parvovirus 4 in the cerebrospinal fluid (CSF) of two patients presenting with acute encephalitis syndrome in northern India. This is the first report of the Parv4 genome sequence from northern India.
Dengue is a common vector-borne disease of tropical and subtropical areas, affecting about 50 mil... more Dengue is a common vector-borne disease of tropical and subtropical areas, affecting about 50 million people each year and exposing more than 2.5 billion people at risk. Dengue virus (DENV), responsible for the disease, belongs to family Flaviviridae, genus Flavivirus and has four distinguished serotypes (DENV 1, DENV 2, DENV 3, and DENV 4). The main vector of the virus is Aedes aegypti mosquito. Water logging increases mosquito density and an increase in vector-borne diseases (such as dengue, malaria, West Nile fever) through the expansion in the number and range of vector habitats. Infection with any of the DENV serotypes may be asymptomatic in the majority of cases or may result in a wide spectrum of clinical symptoms, ranging from a mild flu-like syndrome (known as dengue fever [DF]) to the most severe forms of the disease; Dengue Hemorrhagic Fever/ Dengue Shock Syndrome (DHF/DSS). Recently in 2008, WHO has revised its classification and divides the clinical illness to non-severe dengue, dengue ± warning signs, and severe dengue. Pathogenesis of severe dengue disease is very complex and not properly elucidated. Timely diagnosis of the disease is the key to control it. Routine diagnosis largely depends upon NS1 antigen, IgM, and genome detection by different methods. Confirmatory diagnosis is by cell culture method, the most important of which is C6/36 mosquito cell line or intrathoracic inoculation of mosquito. Till date, no effective vaccine is available in the market. Different approaches are being used to develop various formulations of vaccine, but are of not much use. Alternative vaccine strategies in the form of small interfering RNA (siRNA) and micro RNA (miRNA) seem to be
Dengue is one of the most important mosquito-borne viral diseases that are relentlessly spreading... more Dengue is one of the most important mosquito-borne viral diseases that are relentlessly spreading in newer areas in the tropical and subtropical regions of the World. In last fifty years, in spite of intensive and extensive investigations, pathogenesis of dengue is still not clearly understood. Recently, the research focus is on studying the role of intracellular events in pathogenesis of viral infections. Entry of virion in the host cell is followed by quick succession of events, unfolded protein response, lipid bodies and lipophagy, endoplasmic reticulum stress and recent demonstration of autophagy. The turbulence caused by these events may result in clearance of the virus/enhanced replication and survival of the host cell/apoptosis. Both, increased virus load and apoptosis of host cell may have pathological effects on the host. In the present review, we have summed up the role of various intracellular events in viral infections with special emphasis on Dengue virus infection.
The pandemic H1N1 strain of Influenza A virus [A(H1N1)pdm09] is now well adapted in human populat... more The pandemic H1N1 strain of Influenza A virus [A(H1N1)pdm09] is now well adapted in human populations. However, it is still causing sporadic outbreaks worldwide with different severity. The present study was planned to understand the genetic diversity (based on the HA1 gene) of influenza A(H1N1)pdm09 strains circulating during the post pandemic period. The HA1 gene was selected because the HA1 protein is immunogenic, functions as a receptor binding site and indirectly affects transmission and pathogenicity of virus. A total of 2,818 cases were enrolled. Nasal/throat swabs from all cases were tested by one-step real time PCR for detection of influenza virus types and subtypes according to the CDC protocol. Of these, 134 cases were A(H1N1)pdm09 positive, 34 of which were screened for HA1 gene (position 434-905) sequencing (Big-Dye terminator using 3130 ABI, Genetic analyzer). Molecular and phylogenetic analysis was performed using PhyML approach (v. 3.0). All A(H1N1)pdm09 positive and negative cases were clinically characterized. Phylogentically, all Lucknow strains (n = 33) except one fall with the clade seven reference strain. One strain showed 99.9% similarities with clade one reference strain A/California/07/2009. In mutational analysis, 33 strains had the S220T mutation, which is at an antigenic site and characteristic of clade seven along with few minor mutations; K180I/T/Q, V190I, S200P, S202T, A203T, A214T, S220T, V251I, and A273T. These results suggest that clade seven was the most widely circulating clade in Lucknow and A(H1N1)pdm09 cases showed mild clinical symptoms as compared to A(H3N2) or influenza B cases.
A comparatively newly discovered human metapneumovirus (HMPV) has emerged as an important cause o... more A comparatively newly discovered human metapneumovirus (HMPV) has emerged as an important cause of severe acute respiratory illness (SARI), second only to respiratory syncytial virus (RSV). RSV and HMPV taxonomically belong to same family and subfamily, and their clinical presentation and seasonal distribution are also seemed to be indistinguishable. Present study was planned to know the epidemiology and prevalence of HMPV and RSV in patients presented as SARI in a tertiary care hospital. Nasopharyngeal aspirate of 440 patients fulfilling World Health Organization criteria of SARI, enrolled during a 2-year study period, were collected and tested for the presence of RSV, HMPV and their subtypes A and B by real time polymerase chain reaction along with other respiratory viruses, viz influenza A, B, parainfluenza 1, 2, 3, 4, adenovirus, measles virus and bocavirus. The demographic details, clinical profile, underlying diseases, clinical diagnosis at the time of admission and seasonal distribution were studied and analyzed statistically. Overall positivity of RSV was 14.3% (24.68% in &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;5 years) and of HMPV was 3.63% (5.1% in &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;5 years and 5.08% in 6-12 years). Among RSV, subtype A (89%), and among HMPV, genotype B (68.8%) were predominating. Adults having underlying chronic obstructive pulmonary disease were more prone to acquire RSV and HMPV infections. RSV and HMPV positivity was restricted to winter season. We are reporting replacement of RSV with HMPV in this population. HMPV has emerged as an important cause of SARI in children…
International Journal of Research and Development in Pharmacy & Life Sciences, Oct 1, 2018
Dengue virus is mosquito-borne virus that manifests itself in human infections with dengue fever ... more Dengue virus is mosquito-borne virus that manifests itself in human infections with dengue fever (DF) to dengue hemorrhagic fever (DHF). DHF can lead to development of dengue shock syndrome (DSS). RNAi works by silencing the target gene expression using siRNA. Hence there arises an urgent need to design potential siRNA against the target sequence of dengue virus gene to control its replication and pathogenicity. This study is aimed to predict self-potential designed siRNA-based therapeutics that might be used for the treatment of dengue virus infection. The prediction of potential siRNA was done by using various computational tools as searching target sequences, multiple sequences alignment, secondary structure prediction, siRNA-target sequence interaction prediction and finally the evaluation of effectiveness of predicted siRNA. Ten pair of potential siRNAs were predicted and designed rationally for silencing five target genes (Capsid, CprM, NS1, NS3 and NS5) of dengue virus used in the study through RNAi technology. The outcomes of this study are ten pair of potential siRNA molecules which might be used as a potential antiviral RNA based therapeutics to suppress the dengue virus replication.
International journal of innovation and scientific research, 2015
Chronic obstructive pulmonary disease is the leading cause of fatality. The course of COPD is fol... more Chronic obstructive pulmonary disease is the leading cause of fatality. The course of COPD is followed by episodes of acute deterioration in respiratory health, referred as 'exacerbations'. Acute exacerbations of COPD contribute substantially to the morbidity and mortality due to number of infectious agents including bacteria, viruses, or both. Therefore, we planned a case control study to know the association of respiratory viruses especially HRSV genotype with acute exacerbation of COPD, if any. This is a prospective case-control study with two groups of patients (AECOPD and stable COPD). Nasopharyngeal aspirate were tested for the detection of Human Respiratory Syncytial Virus; Influenza Viruses; Human Metapneumovirus; Adeno Virus; Human Boca Virus and Parainfluenza Virus 1,2,3,4 by real time PCR. Respiratory viruses are more often found in case group (AECOPD patients) 45/ 234 patients (19.23%) than in control group (stable COPD), 8/100 patients (8%; P=0.0330). In case gr...
The study was conducted to analyze changes in HA and NA genes of pandemic pH1N1 strains, collecte... more The study was conducted to analyze changes in HA and NA genes of pandemic pH1N1 strains, collected from North Indian tertiary care hospital during 2015 and studied the nucleotide change since 2009. Differences in clinical features of cases positive and negative for influenza viruses were also studied. All samples referred for H1N1 testing, were tested for influenza viruses. Randomly selected 25 strains of pH1N1 were studied for nucleotide sequences of HA & NA gene. The nucleotide changes occurring since 2009 were studied by mutational and phylogenetic analysis. Clinical details of cases were recorded and analysed. A total of 3319 cases of acute respiratory infections (ILI/SARI) were tested for influenza viruses during Jan to April 2015, of which 815 cases tested positive for pH1N1. Nucleotide variation of 2015 strains, from influenza A/California/07/2009 strain at HA1 and NA1 gene was 1.9% and 3.8% respectively. Both HA1 and NA1 coding sequence showed eight mutations. Four of HA1(K1...
Genomic variability makes Influenza A virus (IAV) 'the least susceptible' to existing vaccines or... more Genomic variability makes Influenza A virus (IAV) 'the least susceptible' to existing vaccines or anti-influenza drugs. siRNA targeting viral gene silents the gene by cleaving mRNA. Present study aimed to develop siRNA targeting polymerase basic 1 (PB1) gene and to validate its efficiency in vitro. siRNA was designed rationally, targeting the most conserved region of PB1 gene of IAV strains. Total 147 strains worldwide and 42 Indian strains, when aligned, showed seven sets of conserved regions (> 30 bp stretch and < 5% mismatches). To choose the most efficient siRNA, three levels screening method was developed. Finally one pair of siRNA was chosen due to its unique position in conserved region. siRNA efficacy was confirmed in vitro on Madin Darby Canine Kidney (MDCK) cell line propagating two clinical isolates i.e. Influenza A/H3N2 [A/India/LKO864/ 2011(H3N2)] and Influenza A/pandemicH1N1 [A/India/LKO2151/2012(H1N1)]. The longest ORF was targeted by the selected siRNA, which showed 57 % inhibition in replication of Influenza A/pdmH1N1 and 60.6 % inhibition in replication of Influenza A/H3N2 at 72 hpi and 48 hpi respectively on MDCK cell line. This study shows that siRNA targeting PB1 may be moderately effective in controlling IAV replication so can be used as anti-IAV therapeutic agent.
Multiplex RT-PCR assays are widely used tools for detection of hepatitis viruses, but none of the... more Multiplex RT-PCR assays are widely used tools for detection of hepatitis viruses, but none of them provide quality check of sample. In the present study we developed a single-step triplex real-time polymerase chain reaction (PCR) assay for detection of Hepatitis B Virus (HBV) and Hepatitis C Virus (HCV) with sample quality check, by using β-actin as housekeeping gene. The primers and probes were self-designed and assay was standardized. Assay was also destined to quantitate copy numbers of HBV and HCV. This novel assay was sensitive, specific, and reproducible for detection of HBV and HCV in serum/plasma. The assay also detected all genotypes of HBV and HCV. The detection limit was 60IU/mL for HBV and 20IU/mL for HCV. This assay is the first assay developed on single-step platform for nucleic acid detection of HBV and HCV with an extra edge over all other assays by providing inbuilt check for quality of sample.
Hepatitis B virus (HBV) is among the most common causes of liver cirrhosis. We report the full-ge... more Hepatitis B virus (HBV) is among the most common causes of liver cirrhosis. We report the full-genome sequences of seven molecular clones of HBV genotype A, amplified from an HBV-infected North Indian patient. This is probably the first report of the HBV genome sequencing using Ion Torrent from India.
The genomic variability of Influenza A virus (IAV) makes it difficult for the existing vaccines o... more The genomic variability of Influenza A virus (IAV) makes it difficult for the existing vaccines or anti-influenza drugs to control. The siRNA targeting viral gene induces RNAi mechanism in the host and silent the gene by cleaving mRNA. In this study, we developed an universal siRNA and validated its efficiency in vitro. The siRNA was designed rationally, targeting the most conserved region (delineated with the help of multiple sequence alignment) of M gene of IAV strains. Three level screening method was adopted, and the most efficient one was selected on the basis of its unique position in the conserved region. The siRNA efficacy was confirmed in vitro with the Madin Darby Canine Kidney (MDCK) cell line for IAV propagation using two clinical isolates i.e., Influenza A/H3N2 and Influenza A/pdmH1N1. Of the total 168 strains worldwide and 33 strains from India, 97 bp long (position 137-233) conserved region was identified. The longest ORF of matrix gene was targeted by the selected si...
International Journal of Applied Sciences and Biotechnology, 2014
Introduction: In spite of the discovery of viral culture technology about a century ago, its appl... more Introduction: In spite of the discovery of viral culture technology about a century ago, its application in diagnostic labs is being used since 1970s. It served as the "gold standard" for virus detection for long. In recent years, rapid, technically less challenging, sensitive and highly specific viral identification is possible by molecular tools. Hence, the purpose of this study was to analyze the importance of real time PCR over virus culture in diagnosis of Influenza virus infections, the biggest viral challenge of present India, a developing country, so that prompt and correct diagnosis can help physicians as well as the policy makers to control the virus spread. Objective: To study the feasibility of real time PCR vis a vis viral culture technique and evaluate the utility of these methods for laboratory diagnosis of Influenza virus infections. Methodology: The study was conducted in Grade I Virology Diagnostic laboratory, Dept of Microbiology, KGMU, Lucknow. We used ...
During the post influenza pandemic period, continuous surveillance of influenza virus and its sub... more During the post influenza pandemic period, continuous surveillance of influenza virus and its subtypes is mandatory to help the policy makers to take effective and appropriate decisions. Therefore, this study was planned to determine the pattern of influenza virus activity in context to various meteorological and clinical parameters in and around Lucknow, Uttar Pradesh, India, during post pandemic period August 2010 - September 2012. Nasal swabs/throat swabs/nasopharyngeal aspirates of 2669 patients were collected. One-step real time PCR for detection of influenza virus was done according to the Centers for Disease Control and Prevention (CDC) protocol. Influenza positivity was 15.8 per cent (423/2669) in symptomatic patients. Of the 423 total positives, 192 (7.2%) were influenza A and 231 (8.7%) were influenza B. Positivity for influenza virus was significantly (P=0.001, OR=2.9, CI=1.9-4.3) higher in patients with Influenza like illness (ILI) (17.4%, 396/2271) than those with sever...
The human respiratory syncytial virus (HRSV) is a community-acquired virus that mainly causes acu... more The human respiratory syncytial virus (HRSV) is a community-acquired virus that mainly causes acute respiratory tract infection in infants and children. HRSV is increasingly recognized as an important nosocomial pathogen causing morbidity in immunocompromised patients. Here, we are reporting a hospital outbreak of HRSV during summer in children receiving chemotherapy for haematological malignancies. Prompt detection and timely preventive measures could abort the devastating outbreak. In the month of June 2013, seven children from paediatric oncology Ward at King George Medical University presented with respiratory signs and symptoms in span of 3 days. Nasal and throat swabs of children were tested for the presence of HRSV, human metapneumovirus and their subtypes A and B, influenza A and B, measles, parainfluenza virus (1, 2, 3 and 4), adenovirus, and human bocavirus by real-time polymerase chain reaction. A segment of F gene of HRSV was amplified and sequenced for phylogenetic anal...
Human parvovirus 4 (Parv4) is a relatively new virus. Association of this virus with any human di... more Human parvovirus 4 (Parv4) is a relatively new virus. Association of this virus with any human disease is yet to be established. We detected human parvovirus 4 in the cerebrospinal fluid (CSF) of two patients presenting with acute encephalitis syndrome in northern India. This is the first report of the Parv4 genome sequence from northern India.
Dengue is a common vector-borne disease of tropical and subtropical areas, affecting about 50 mil... more Dengue is a common vector-borne disease of tropical and subtropical areas, affecting about 50 million people each year and exposing more than 2.5 billion people at risk. Dengue virus (DENV), responsible for the disease, belongs to family Flaviviridae, genus Flavivirus and has four distinguished serotypes (DENV 1, DENV 2, DENV 3, and DENV 4). The main vector of the virus is Aedes aegypti mosquito. Water logging increases mosquito density and an increase in vector-borne diseases (such as dengue, malaria, West Nile fever) through the expansion in the number and range of vector habitats. Infection with any of the DENV serotypes may be asymptomatic in the majority of cases or may result in a wide spectrum of clinical symptoms, ranging from a mild flu-like syndrome (known as dengue fever [DF]) to the most severe forms of the disease; Dengue Hemorrhagic Fever/ Dengue Shock Syndrome (DHF/DSS). Recently in 2008, WHO has revised its classification and divides the clinical illness to non-severe dengue, dengue ± warning signs, and severe dengue. Pathogenesis of severe dengue disease is very complex and not properly elucidated. Timely diagnosis of the disease is the key to control it. Routine diagnosis largely depends upon NS1 antigen, IgM, and genome detection by different methods. Confirmatory diagnosis is by cell culture method, the most important of which is C6/36 mosquito cell line or intrathoracic inoculation of mosquito. Till date, no effective vaccine is available in the market. Different approaches are being used to develop various formulations of vaccine, but are of not much use. Alternative vaccine strategies in the form of small interfering RNA (siRNA) and micro RNA (miRNA) seem to be
Dengue is one of the most important mosquito-borne viral diseases that are relentlessly spreading... more Dengue is one of the most important mosquito-borne viral diseases that are relentlessly spreading in newer areas in the tropical and subtropical regions of the World. In last fifty years, in spite of intensive and extensive investigations, pathogenesis of dengue is still not clearly understood. Recently, the research focus is on studying the role of intracellular events in pathogenesis of viral infections. Entry of virion in the host cell is followed by quick succession of events, unfolded protein response, lipid bodies and lipophagy, endoplasmic reticulum stress and recent demonstration of autophagy. The turbulence caused by these events may result in clearance of the virus/enhanced replication and survival of the host cell/apoptosis. Both, increased virus load and apoptosis of host cell may have pathological effects on the host. In the present review, we have summed up the role of various intracellular events in viral infections with special emphasis on Dengue virus infection.
The pandemic H1N1 strain of Influenza A virus [A(H1N1)pdm09] is now well adapted in human populat... more The pandemic H1N1 strain of Influenza A virus [A(H1N1)pdm09] is now well adapted in human populations. However, it is still causing sporadic outbreaks worldwide with different severity. The present study was planned to understand the genetic diversity (based on the HA1 gene) of influenza A(H1N1)pdm09 strains circulating during the post pandemic period. The HA1 gene was selected because the HA1 protein is immunogenic, functions as a receptor binding site and indirectly affects transmission and pathogenicity of virus. A total of 2,818 cases were enrolled. Nasal/throat swabs from all cases were tested by one-step real time PCR for detection of influenza virus types and subtypes according to the CDC protocol. Of these, 134 cases were A(H1N1)pdm09 positive, 34 of which were screened for HA1 gene (position 434-905) sequencing (Big-Dye terminator using 3130 ABI, Genetic analyzer). Molecular and phylogenetic analysis was performed using PhyML approach (v. 3.0). All A(H1N1)pdm09 positive and negative cases were clinically characterized. Phylogentically, all Lucknow strains (n = 33) except one fall with the clade seven reference strain. One strain showed 99.9% similarities with clade one reference strain A/California/07/2009. In mutational analysis, 33 strains had the S220T mutation, which is at an antigenic site and characteristic of clade seven along with few minor mutations; K180I/T/Q, V190I, S200P, S202T, A203T, A214T, S220T, V251I, and A273T. These results suggest that clade seven was the most widely circulating clade in Lucknow and A(H1N1)pdm09 cases showed mild clinical symptoms as compared to A(H3N2) or influenza B cases.
A comparatively newly discovered human metapneumovirus (HMPV) has emerged as an important cause o... more A comparatively newly discovered human metapneumovirus (HMPV) has emerged as an important cause of severe acute respiratory illness (SARI), second only to respiratory syncytial virus (RSV). RSV and HMPV taxonomically belong to same family and subfamily, and their clinical presentation and seasonal distribution are also seemed to be indistinguishable. Present study was planned to know the epidemiology and prevalence of HMPV and RSV in patients presented as SARI in a tertiary care hospital. Nasopharyngeal aspirate of 440 patients fulfilling World Health Organization criteria of SARI, enrolled during a 2-year study period, were collected and tested for the presence of RSV, HMPV and their subtypes A and B by real time polymerase chain reaction along with other respiratory viruses, viz influenza A, B, parainfluenza 1, 2, 3, 4, adenovirus, measles virus and bocavirus. The demographic details, clinical profile, underlying diseases, clinical diagnosis at the time of admission and seasonal distribution were studied and analyzed statistically. Overall positivity of RSV was 14.3% (24.68% in &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;5 years) and of HMPV was 3.63% (5.1% in &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;5 years and 5.08% in 6-12 years). Among RSV, subtype A (89%), and among HMPV, genotype B (68.8%) were predominating. Adults having underlying chronic obstructive pulmonary disease were more prone to acquire RSV and HMPV infections. RSV and HMPV positivity was restricted to winter season. We are reporting replacement of RSV with HMPV in this population. HMPV has emerged as an important cause of SARI in children…
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