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Biomedical subjects

S K Bandyopadhyay

Publications and source records attributed to S K Bandyopadhyay.

At least 19 recordsLinked to original sources

Development and characterization of a stable vero cell line constitutively expressing Peste des petits ruminants virus (PPRV) hemagglutinin protein and its potential use as antigen in enzyme-linked immunosorbent assay for serosurveillance of PPRV.

We developed and characterized a stable Vero cell line constitutively expressing Peste des petits ruminants virus (PPRV) hemagglutinin (H) protein and assessed its potential use as diagnostic antigen in enzyme-linked immunosorbent assay (ELISA). PPRV H gene of the vaccine strain (Sungri-96) was amplified by reverse transcription (RT)-PCR, cloned into a eukaryotic expression vector (pTarget), and subsequently transfected and expressed in Vero cells. A stable Vero cell line was developed after 20 repeated passages by using G418 antibiotic selection pressure (400 to 600 microg/ml). The integration of PPRV H gene in the Vero cell genome and its genomic transcription were confirmed by PCR and RT-PCR assays, respectively, and the 70-kDa PPRV H protein was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. The recombinant protein reacted specifically with PPRV anti-H neutralizing monoclonal and polyclonal antibody in competitive, sandwich, and indirect ELISA, respectively, indicating that the native form of the protein was expressed. Evaluation of the protein in competitive ELISA and indirect ELISA vis a vis whole virus was done using 306 and 146 goat field serum samples, respectively; comparable results were obtained with high degrees of relative diagnostic specificity (93.53% and 100%, respectively) and sensitivity (99.04% and 79.16%, respectively). This study shows that the PPRV H protein could be a sustainable source of safe antigen in countries of nonendemicity without the need to handle infectious virus for serodiagnosis.

Animals↗

Sequence analysis of the non-structural 3A and 3C protein-coding regions of foot-and-mouth disease virus serotype Asia1 field isolates from an endemic country.

A total of 18 foot-and-mouth disease virus (FMDV) serotype Asia1 field isolates belonging to two different lineages (including the divergent group) as delineated earlier in VP1-based phylogeny were sequenced in the non-structural 3A and 3C protein-coding regions. The phylogenetic trees representing the regions coding for the non-structural proteins were very similar to that of the structural VP1 protein-coding region. Phylogenetic comparison at 3C region revealed clustering of Asia1 viruses with the isolates of serotypes O, A and C in the previously identified clade. Comparison of amino acid sequences identified lineage-specific signature residues in both the non-structural proteins. Overall analysis of the amino acid substitutions revealed that the 3A coding region was more prone to amino acid alterations than 3C region.

Animals↗

One-step multiplex RT-PCR assay for the detection of peste des petits ruminants virus in clinical samples.

A single-tube one-step multiplex RT-PCR was standardized to amplify both 337 bp and 191 bp fragments of N and M genes of peste des petits ruminants virus (PPRV), respectively, and only a 337 bp fragment of N gene of Rinderpest virus (RPV). The RT-PCR using purified viral RNA was easily adopted for direct detection of PPRV in clinical field samples and its differentiation from RPV. The amplified N and M gene products were confirmed to be PPRV- and RPV-specific by their size in 1.5% agarose gel and restriction analysis. In the assay, the Qiagen one-step RT-PCR kit containing the Ominiscript and Sensiscript reverse transcriptases and Hot star Taq DNA polymerase was utilized. The sensitivity of the assay was found to be 100 fg of PPRV RNA. Compared with a two-step assay, the one-step assay is easier and time-saving as it requires just a single buffer for both reactions, reverse transcription (RT) and PCR. In experimentally infected goats, PPRV was detectable by the one-step RT-PCR in nasal and ocular swabs 7-17 days post infection (p.i.). and in oral swabs 7-15 days p.i. Out of 32 clinical field samples tested, 18 were positive by sandwich ELISA (S-ELISA), while 22 were positive by the one-step RT-PCR.

Animals↗

Sequence analysis of the nucleoprotein gene of Asian lineage peste des petits ruminants vaccine virus.

The complete nucleotide sequence of the nucleocapsid (N) protein of the peste-des-petits ruminants vaccine virus (PPRV Sungri/96) belonging to the Asian lineage was determined. The gene was 1692 nucleotides in length and encoded a polypeptide of 525 amino acids. The PPRV Sungri/96 N gene has a nucleotide homology of 92% for PPRV Nigeria 75/1 to 55.5% for canine distemper virus. At amino acid level the homology was 94.1% with PPRV Nigeria 75/1, while with other morbilliviruses, PPRV Sungri/96 had only 71.4-64.9% amino acid identity. The phosphorylation prediction reveals eight conserved sites across morbilliviruses, whereas in the C-terminal portion of the protein the sites are not conserved. Phylogenetic analysis of different N proteins of morbilliviruses revealed five well-defined clusters as observed previously. To the best of our knowledge this is the first report describing the nucleocapsid gene sequence of PPRV Indian isolate.

Amino Acid Sequence↗

Sequence analysis of the haemagglutinin and fusion protein genes of peste-des-petits ruminants vaccine virus of Indian origin.

The amino acid composition of the two surface proteins of peste-des-petits ruminants vaccine virus belonging to lineage four from India were deduced from the nucleotide sequence. The fusion (F) protein gene of PPRV Sungri/96 is 2405 nucleotides long and in relation to the length, it is 80 nucleotides longer than that of PPRV Nigeria/75/1 which are found to be present at the 5'UTR of this virus. The complete F gene alignment with other morbillivirus reveals a homology of 89% with PPRV/Nigeria/75/1 and 48-51% with other morbilliviruses. The F protein of PPRV Sungri/96 exhibited characteristics similarity to those of other morbillivirus F proteins. The overall amino acid similarity with its counterpart PPRV Nigeria/75/1 was 96%; with other morbilliviruses it is 65-74%. The PPRV Sungri/96 haemagglutinin (H) protein gene is 1954 nucleotides long and showed a sequence homology of 90.7% with PPRV/Nigeria/75/1 and with other morbilliviruses it ranged from 33% to 45%. At amino acids level, PPRV Sungri/96 showed a homology of 92.3% with PPRV/Nigeria/75/1 and 34-49% with other morbilliviruses. The phylogenetic tree constructed for F and H gene reveals four separate groups which is very similar to that found in other genes. To the best of our knowledge this is the first report describing the F and H genes of an Indian isolate.

Amino Acid Sequence↗

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.

Amino Acid Sequence↗

Isolated massive thyroid metastasis in lung cancer.

Metastasis to the thyroid gland is rare despite its rich vascular supply. Among the pulmonary malignancies metastasising to the thyroid, adenocarcinomas are the commonest. The appearance of metastatic disease in lung carcinoma indicates a poor prognosis and the average survival is two months. We report a 62-year-old woman with squamous cell carcinoma of lung metastatic to the thyroid that produced massive enlargement of the gland. The appearance of the secondary preceded the diagnosis of the primary malignancy by a few months. Ultimately, the patient succumbed to her disease.

Carcinoma, Squamous Cell↗

Melnick- needles osteodysplasty presenting with quadriparesis.

Melnick-Needles syndrome or osteodysplasty, a monogenic heritable bone dysplasia, is characterized by a typical facies and characteristic radiological findings. Less than 70 well-documented cases have been reported in literature; most of them were sporadic. We report the first case from Eastern India in an adolescent male, who had cranio-vertebral junction anomalies and presented with spastic quadriparesis at the age of 13 years.

Adolescent↗

The M and N genes-based simplex and multiplex PCRs are better than the F or H gene-based simplex PCR for Peste-des-petits-ruminants virus.

Nucleocapsid (N), matrix (M) and hemagglutinin (H) genes-based simplex PCRs and an N and M genes-based multiplex PCR were developed for detection of Peste-des-petits-ruminants virus (PPRV). The M gene PCR was the most sensitive, followed by N, H and an already described fusion (F) gene PCRs, as they could detect the virus in samples with titers of 101, 102, 104and 105 TCID50/ml, respectively. The multiplex PCR was as sensitive as the M gene PCR, but it had the advantage of differentiating PPRV from Rinderpest virus (RPV).

Electrophoresis, Agar Gel↗

A novel genetic lineage differentiating RT-PCR as a useful tool in molecular epidemiology of foot-and-mouth disease in India.

Comparison of nucleotide sequences at the VP1 coding region of foot-and-mouth disease serotype Asia1 viruses from India has revealed two genetic lineages with emergence of a genetically divergent group in recent years. In this study a simple, fast, relatively costeffective multi-primer RT-PCR assay to differentiate genetic lineages of type Asia1 viruses was developed. Efforts were made in the design of novel lineage-specific primers and in optimization of the multi-primer assay protocol in conjunction with the use of the serotype specific primer for confirmation of serotype Asia1 virus. This assay promises to be an effective tool in molecular epidemiological investigation of FMD in the country.

Capsid Proteins↗