Production and characterization of monoclonal antibodies to Newcastle Disease Virus.

Newcastle Disease (ND) is one of the major causes of economic loss in the poultry industry. Newcastle Disease Virus (NDV) is a single-stranded, negative-sense enveloped RNA virus (Fam. Paramyxoviridae; Order Mononegavirales). In the present study three monoclonal antibodies (MAbs) were produced by polyethyleneglycol (PEG)-mediated fusion of lymphocytes sensitized to NDV Bareilly strain and myeloma cells. NDV possesses ability to agglutinate erythrocytes of avian species. All the three MAbs designated as 2H7, 3E9 and 3G6 caused hemagglutination inhibition of NDV by specifically binding to NDV. The reactivity for all the 3 MAbs on indirect ELISA was found to be significantly higher than the antibody and antigen controls. On flowcytometry of HeLa cells infected with NDV using the MAbs as primary antibodies, there was a significant difference in the percentage of cells showing positive fluorescence compared to the mock control. One of the MAbs (3E9) was found to react with hemagglutinin-neuraminidase (HN) protein on western blot.

Phylogenetic analysis of glycoprotein B gene sequences of bovine herpesvirus 1 isolates from India reveals the predominance of subtype 1.1.

AIM: This study was conducted for the isolation and molecular characterization of bovine herpesvirus 1 (BoHV-1) isolated from the nasal and vaginal swabs collected from naturally infected cattle showing clinical symptoms of the respiratory disease. MATERIALS AND METHODS: Isolation of BoHV-1 virus performed on clinical samples collected from 65 cattle from five states of India. The BoHV-1 isolates were further confirmed by polymerase chain reaction (PCR) using primers specific for glycoprotein B (gB) genomic region. PCR amplification was performed using previously published gB gene-specific primer pairs. gB PCR amplicons obtained from all isolates were sequenced, and phylogenetic analysis was performed using software. RESULTS: A total of 12 samples were found positive in cell culture isolation. 11 isolates showed the visible cytopathic effect on Madin-Darby bovine kidney after 72 h. Partial sequence analysis of gB gene of all isolates revealed 99.0-100% homology between them. All isolates showed 99.2-99.8% homology with Cooper stain. CONCLUSION: BoHV-1.1 is the predominant circulating subtype of BoHV in India, and all isolates have homology with Cooper stain.

Characterization and evaluation of apoptotic potential of double gene construct pVIVO.VP3.NS1.

Viral gene oncotherapy, targeted killing of cancer cells by viral genes, is an emerging non-infectious therapeutic cancer treatment modality. Chemo and radiotherapy in cancer treatment is limited due to their genotoxic side effects on healthy cells and need of functional p53, which is mutated in most of the cancers. VP3 (apoptin) of chicken infectious anaemia (CIA) and NS1 (Non structural protein 1) of Canine Parvovirus-2 (CPV-2) have been proven to have oncolytic potential in our laboratory. To evaluate oncolytic potential of VP3 and NS1 together these genes needed to be cloned in a bicistronic vector. In this study, both these genes were cloned and characterized for expression of their gene products and its apoptotic potential. The expression of VP3 and NS1 was studied by confocal microscopy and flowcytometry. Expression of VP3 and NS1 in pVIVO.VP3.NS1 transfected HeLa cells in comparison to mock transfected cells indicated that the double gene construct expresses both the products. This was further confirmed by flowcytometry where there was increase in cells expressing VP3 and NS1 in pVIVO.VP3.NS1 transfected group in comparison with the mock control group. The apoptotic inducing potential of this characterized pVIVO.VP3.NS1 was evaluated in human cervical cancer cell line (HeLa) by DNA fragmentation assay, TUNEL assay and Hoechst staning. This double construct was observed to induce apoptosis in HeLa cells.

Development of dog mammary tumor xenograft in immunosuppressed Swiss albino mice.

Development and study of dog mammary tumour xenograft in immunosuppressed Swiss Albino Mice adds a new dimension in cancer research as dog tumors have many similarities with human tumors regarding progression, histopathology, molecular mechanism, immune response and therapy. Failure of the immune system to recognize and eliminate cancer cells leads to cancer progression and the fight between immune cells and cancer cells has a great role in understanding the mechanism of cancer progression and elimination. Rejection and acceptance of tumour xenograft depends on efficiency of CD4+, CD8+ and NK cell populations. In the present investigation, dog mammary tumor xenograft in cyclosporine-A and gamma-irradiated, immunosuppressed Swiss Albino mice was developed and the immune cell status of graft accepted and rejected mice was assessed. It was observed that all the major immune cells (CD4+, CD8+ and NK cells) play an equal role in tumour rejection.

Non-Structural protein 1 (NS1) gene of Canine Parvovirus-2 regresses chemically induced skin tumors in Wistar rats.

The Non-Structural protein 1 of Canine Parvovirus-2 (CPV2.NS1) plays a major role in viral cytotoxicity and pathogenicity. CPV2.NS1 has been proven to cause apoptosis in HeLa cells in vitro in our laboratory. Here we report that CPV2.NS1 has no toxic side effects on healthy cells but regresses skin tumors in Wistar rats. Histopathological examination of tumor tissue from CPV2.NS1 treated group revealed infiltration of mononuclear and polymorphonuclear cells with increased extra cellular matrix, indicating signs of regression. Tumor regression was also evidenced by significant decrease in mitotic index, AgNOR count and PCNA index, and increase in TUNEL positive apoptotic cells in CPV2.NS1 treated group. Further, CPV2.NS1 induced anti-tumor immune response through significant increase in CD8(+) and NK cell population in CPV2.NS1 treated group. These findings suggest that CPV2.NS1 can be a possible therapeutic candidate as an alternative to chemotherapy for the treatment of cancer.

Canine parvovirus type 2a (CPV-2a)-induced apoptosis in MDCK involves both extrinsic and intrinsic pathways.

The canine parvovirus type 2 (CPV-2) causes an acute disease in dogs. It has been found to induce cell cycle arrest and DNA damage leading to cellular lysis. In this paper, we evaluated the apoptotic potential of the "new CPV-2a" in MDCK cells and elucidated the mechanism of the induction of apoptosis. The exposure of MDCK cells to the virus was found to trigger apoptotic response. Apoptosis was confirmed by phosphatidylserine translocation, DNA fragmentation assays, and cell cycle analysis. Activation of caspases-3, -8, -9, and -12 and decrease in mitochondrial potential in CPV-2a-infected MDCK cells suggested that the CPV-2a-induced apoptosis is caspase dependent involving extrinsic, intrinsic, and endoplasmic reticulum pathways. Increase in p53 and Bax/Bcl2 ratio was also observed in CPV-2a-infected cells.

Apoptosis induced by NS1 gene of Canine Parvovirus-2 is caspase dependent and p53 independent.

Apoptosis is programmed cell death that normally occurs during development and aging in multicellular animals. Apoptosis also occurs as a defense mechanism against disease or harmful external agents. It can be initiated by a variety of stimuli including viruses and viral proteins. Canine parvovirus type 2 (CPV-2) that causes acute disease in dogs has been found to induce cell cycle arrest and DNA damage leading to cellular lysis. Though non structural protein 1 (NS1) of many parvoviruses has been found to be apoptotic, no report on the apoptotic potential of NS1 of CPV-2 (CPV-2.NS1) exists. In this study, we evaluated the apoptotic potential of CPV-2.NS1 in HeLa cells. CPV-2.NS1 has been found to induce apoptosis which was evident through characteristic DNA fragmentation, increase in hypodiploid cell count, phosphatidyl serine translocation and activation of caspase-3. Increase in caspase-3 activity and no change in p53 activity with time in CPV-2.NS1 expressing HeLa cells showed the induction of apoptosis to be caspase dependent and p53 independent.

Comparative assessment of three image reconstruction techniques for image quality and radiation dose in patients undergoing abdominopelvic multidetector CT examinations.

OBJECTIVE: To compare image quality and radiation dose of abdominal CT examinations reconstructed with three image reconstruction techniques. METHODS: In this Institutional Review Board-approved study, contrast-enhanced (CE) abdominopelvic CT scans from 23 patients were reconstructed using filtered back projection (FBP), adaptive statistical iterative reconstruction (ASiR) and iterative reconstruction in image space (IRIS) and were reviewed by two blinded readers. Subjective (acceptability, sharpness, noise and artefacts) and objective (noise) measures of image quality were recorded for each image data set. Radiation doses in CT dose index (CTDI) dose-length product were also calculated for each examination type and compared. Imaging parameters were compared using the Wilcoxon signed rank test and a paired t-test. RESULTS: All 69 CECT examinations were of diagnostic quality and similar for overall acceptability (mean grade for ASiR, 3.9±0.3; p=0.2 for Readers 1 and 2; IRIS, 3.9±0.4, p=0.2; FBP, 3.8±0.9). Objective noise was considerably lower with both iterative techniques (p<0.0001 and 0.0016 for ASiR and IRIS). Recorded mean radiation dose, i.e. CTDI(vol), was 24% and 10% less with ASiR (11.4±3.4 mGy; p<0.001) and IRIS (13.5±3.7 mGy; p=0.06), respectively, than with FBP: 15.0±3.5 mGy. CONCLUSION: At the system parameters used in this study, abdominal CT scans reconstructed with ASiR and IRIS provide diagnostic images with reduced image noise and 10-24% lower radiation dose than FBP. ADVANCES IN KNOWLEDGE: CT images reconstructed with FBP are frequently noisy on lowering the radiation dose. Newer iterative reconstruction techniques have different approaches to produce images with less noise; ASiR and IRIS provide diagnostic abdominal CT images with reduced image noise and radiation dose compared with FBP. This has been documented in this study.

Computational modeling and validation studies of 3-D structure of neuraminidase protein of H1N1 influenza A virus and subsequent in silico elucidation of piceid analogues as its potent inhibitors.

Emergence of the drug resistant variants of the Influenza A virus in the recent years has aroused a great need for the development of novel neuraminidase inhibitors for controlling the pandemic. The neuraminidase (NA) protein of the influenza virus has been the most potential target for the anti-influenza. However, in the absence of any experimental structure of the drug targeting NA protein of H1N1 influenza A virus as zanamivir and oseltamivir, the comprehensive study of the interaction of the drug molecules with the target protein has been missing. Hence in this study a computational 3-D structure of neuraminidase of H1N1 influenza A virus has been developed using homology modeling technique, and the same was validated for its reliability by ProSA web server in term of energy profile & Z scores and PROCHECK program followed by Ramachandran plot. Further, the developed 3-D model had been employed for docking studies with the class of compounds as Piceid and its analogs. In this context, two novel compounds (ChemBank ID 2110359 and 3075417) were found to be more potent inhibitors of neuraminidase than control drugs as zanamivir and oseltamivir in terms of their robust binding energies, strong inhibition constant (Ki) and better hydrogen bond interactions between the protein-ligand complex. The interaction of these compounds with NA protein has been significantly studied at the molecular level.

In vitro expression studies of non structural 1 protein of Canine Parvo virus 2 by polyclonal antiserum raised against CPV2-NS1 protein expressed in Escherichia coli as an antigen.

The canine Parvovirus 2, non-structural 1 (NS1) is a novel candidate tumor suppressor gene. To confirm the expression of the NS1 in HeLa cells after transfection there was a need to raise antiserum against CPV2- NS1. Therefore, this study was carried out to express and purify the recombinant NS1 (rNS1), and characterize the polyclonal serum. CPV2-NS1, complete coding sequence (CDS) was amplified, cloned in pET32a+ and expressed in BL21 (DE3) (pLysS). SDS-PAGE analysis revealed that the expression of the recombinant protein was maximum when induced with 1.5 mM IPTG. The 6 x His tagged fusion protein was purified on Ni-NTA resin under denaturing conditions and confirmed by western blot using CPV2 specific antiserum. The rabbits were immunized with the purified rNS1 to raise anti-NS1 polyclonal antiserum. The polyclonal serum was tested for specificity and used for confirming the expression of NS1 in HeLa transfected with pcDNA.cpv2.ns1 by indirect fluorescent antibody test (IFAT), flow cytometry and western blot. The polyclonal antiserum against NS1 could be very useful to establish functional in vitro assays to explore role of NS1 in cancer therapeutics.

Classical swine fever in the pygmy hog.

The pygmy hog is a rare, small and highly endangered mammal belonging to the Suidae family, and it is presently found only in the Assam state of India. While investigating the cause of death of pygmy hogs housed at a conservation centre for captive breeding and research at Basistha, Assam, it was confirmed that they were susceptible to and died as a result of contracting classical swine fever (CSF), caused by CSF virus (CSFV), which is a highly infectious endemic disease of domestic pigs in India. The post-mortem findings and serum CSFV-specific antibody titres, along with the isolation of CSFV from two pygmy hogs, and further confirmation by CSFV genomic E2 and 5' untranslated region (UTR) gene amplification in PCR (polymerase chain reaction), clearly established the cause of death of the pygmy hogs. Further, on phylogenetic analysis, the pygmy hog CSFV 5' UTR sequences were grouped in the genotype 1.1 cluster of Indian CSFVs, and hence the strains causing infection were closely related to CSFV isolates circulating in domestic pigs. Therefore, the occurrence of CSF in this endangered species may pose a potent threat to their existence unless properly controlled, and thus it needs urgent attention. To the authors' knowledge this is the first report on CSF in pygmy hogs.

5'-UTR-based phylogenetic analysis of Classical swine fever virus isolates from India.

Classical swine fever (CSF) caused by Classical swine fever virus (CSFV) is a globally significant disease of pigs. Genetic typing of CSFV isolates can help in understanding the epidemiology of disease and trace down the source of outbreak. 5'-UTR sequence analysis and subsequent genetic classification of nine CSFV field isolates from India indicated that 3 isolates belonged to genotype 2.1 and were closely related to European CSFV strains. The remaining 6 isolates belonged to genotype 1 that contained old and new strains. However, the genotype 2.1 group consisted of recent field isolates only. The study showed circulation of both genotypes 1 and 2.1 in north-eastern part of India.

Sequence analysis of the S10 gene of six Bluetongue Virus isolates from India.

Bluetongue Virus (BTV) genome segment 10 (S10)-based phylogenetic studies are important in understanding the BTV evolution. S10 gene-based phylogenetic analysis grouped six different BTV isolates (BTV serotype 1, 18 and 23) from India in subclade A1 and showed closer relationship with BT viruses from Mediterranean Basin. Indian BTV serotypes 18 and 23 formed a single cluster distinct from BTV serotype 1 isolates and were evolved from BTV from China, Indonesia and Australia. The overall S10 sequences of BTV isolates from India were largely conserved (>95.7% homology) and were distinct from other BT viruses of the world.

Sequence analysis of VP7 gene of Indian bluetongue virus serotype-23 shows its close phylogenetic relationship to Australian and Chinese serotypes.

Bluetongue, an arthropod borne viral disease of wild and domestic ruminants, causes heavy economic losses throughout the world. In the present study, full-length VP7 gene of Indian bluetongue virus (BTV) serotype 23 was sequenced and compared with prototype strains of BTV reported from different countries. Nucleotide sequence analysis of VP7 gene revealed Indian BTV serotype 23 to have 1154 nucleotides with the deletion of two nucleotides at 3' non-coding region and a unique amino acid change 211S-N. The Indian virus also demonstrated a maximum similarity of 94.2% with Australian serotype 1 and a minimum similarity of 67.4% with Australian serotype 15. However, at deduced amino acid level, it had maximum similarity of 99.7% and a minimum of 82.5% with Chinese serotypes 1, 2 and 4 and Australian serotype 15, respectively. Deduced amino acid sequence analysis of putative receptor binding domain (121-249) revealed all the nine hydrophilic domains to be conserved across the serotypes. Functional motifs present in VP7 protein were also conserved in almost all the BTV serotypes including Indian serotype 23. Phylogenetic analysis based on VP7 gene sequence revealed Indian BTV serotype 23 segregating into a monophyletic group along with Australian serotype 1 and Chinese serotypes 1, 2 and 4, indicating its close evolutionary relationship with these Australian and Chinese serotypes.

Extraction and separation studies of tellurium(IV) with tris-(2-ethyl hexyl) phosphate.

A method is proposed for the extraction of microgram levels of tellurium(IV) from halide media with tris-(2-ethyl hexyl) phosphate dissolved in toluene as extractant. The optimum conditions have been evaluated from a critical study of acid concentration, extractant concentration, period of equilibration and effect of solvent. Tellurium ion from the organic phase is stripped with water and determined spectrophotometrically with stannous chloride. The method affords binary separation of tellurium from copper, bismuth, gold and selenium and is applicable to the analysis of alloy samples and synthetic mixtures. The method is fast, accurate and precise.