Genetic characterization of the glycoprotein G of Chandipura viruses in India with emphasis on an outbreak of 2015.

Chandipura virus (CHPV) is found to be associated with sporadic encephalitis outbreaks in humans in India since 1965. We report here, the investigation of CHPV activity during the period of June-August 2015 in the state of Gujarat, which revealed 24.44% positivity among 45 referred encephalitis cases. Phylogenetic study of the G gene sequences of strains from Gujarat 2015 along with available sequences of additional strains from different geographical locations and isolation years (1965-2015), indicated the relatedness of the 2015 strain to a group of the CHPV prototype strain of 1965 and the earliest outbreak strains of 2003. Analyses of selection pressure in the G gene revealed positively selected sites within the signal peptide region and a putative CHPV epitope. These results indicate a probable role of G protein-based immune selection and underline the need for continued surveillance to monitor genetic and antigenic variations in the CHPV.

Development of a novel rapid micro-neutralization ELISA for the detection of neutralizing antibodies against Chandipura virus.

BACKGROUND: Chandipura virus (CHPV) is a leading cause of acute encephalitis with high mortality in paediatric population in India. A micro-neutralization ELISA (MN-ELISA) assay was developed for the detection of neutralizing antibodies (Nab) against CHPV. This novel method gives read-out in the form of ELISA optical density (OD) values and has a shorter turn-around time (TAT) as compared to the conventional cytopathic effect (CPE)-based neutralization assay (MN-CPE). The assay was developed using an Indian strain of CHPV. During the development of the assay different parameters such as cell count, dilution of primary and secondary antibodies and time point for the test termination were optimized. The new and conventional assays were run in parallel where known positive and negative human serum samples were used as test controls. The conventional MN-CPE was terminated at 48h post-infection (p.i.) and stained with Amido black, while in the new assay, MN-ELISA was terminated at pre-determined 18h p.i. and the infected cells were fixed with acetone, followed by in-situ ELISA. Results of both the assays were compared. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the new test was 100% when compared with the conventional MN-CPE method as a 'gold standard'. The MN-ELISA showed two-fold higher antibody titer in one sample and one sample was additionally positive than MN-CPE ELISA. CONCLUSION: The MN-ELISA is rapid, more sensitive and read-out of results is by measurement of OD, which could be more accurate than manual observation of reduction in CPE. This novel test could be used as an alternative to the conventional MN-CPE based assay in sero-surveillance and in future vaccine studies.

A study of tubercular lymphadenitis: a comparison of various laboratory diagnostic modalities with a special reference to tubercular polymerase chain reaction.

OBJECTIVE: The purpose of our study was to compare various laboratory diagnostic methods, namely histopathological examination, Ziehl-Neelsen (ZN) stain, AFB culture by conventional Lowenstein-Jensen (LJ) method and fluorescence-based mycobacterial growth indicator tube (MGIT) technique and polymerase chain reaction (PCR) in clinically suspected cases of tubercular lymphadenitis. MATERIALS AND METHODS: A total of 65 lymph nodes biopsied from patients clinically suspected of having tubercular lymph nodes were included. Specimens were processed for AFB culture after NaOH-NALC concentration and inoculation on LJ medium and using the MGIT system. PCR was performed on all specimens using a commercial nested PCR kit targeting IS6110 insertion element of Mycobacterium tuberculosis complex. All lymph node specimens were subjected to histopathological examination. RESULTS: Of the 65 lymph nodes, 37 (56.9%) were positive on MGIT culture and 45 (69.2%) were positive by PCR. Histopathology showed maximum sensitivity (96%) but with compromised specificity (78.5%). PCR showed 90.1% sensitivity and 100% specificity. The mean turnaround time for mycobacterial growth in smear negative specimens was 30 days determined by LJ and 20 days by MGIT techniques. CONCLUSION: PCR is a rapid and useful method for diagnosis of TB lymphadenitis and definitely increases the positive predictive value of a positive histopathology report. MGIT is better than LJ culture as regards time to positivity and higher yield.

Rapid ELISA for the diagnosis of rotavirus.

BACKGROUND & OBJECTIVES: Rotavirus is the major cause of gastroenteritis in infants and young children all over the world. The objective of the study was to develop a rapid ELISA for the diagnosis of rotavirus infection in children hospitalised with diarrhoea. METHODS: Immune serum was raised in rabbits by inoculating semipurified rotavirus, SA-11 strain. Immunoglobulins were conjugated to horse radish peroxidase and a rapid ELISA for rotavirus diagnosis was developed. The rapid ELISA was compared with routine ELISA, developed earlier at NIV. RESULTS: Of the 155 faecal samples from patients with diarrhoea, 96 were positive by rapid ELISA and 95 in routine NIV ELISA. OD values were higher in rapid ELISA. The rapid ELISA takes only 4 h to complete. INTERPRETATION & CONCLUSION: Rotavirus diagnosis by rapid ELISA is simple and easy to perform. This may lead to a significant reduction in the unnecessary usage of antibiotics, which cannot control infection due to rotavirus. This technology is being commercialized.

Cell-cell adhesion in human fibroblasts requires calcium signaling.

In connective tissues, intercellular adhesion is essential for tissue morphogenesis, development and wound healing. However, the signaling mechanisms initiated by cell-cell adhesion in fibroblasts and that regulate it are not known. In this study we tested the hypothesis that intracellular calcium signaling is required to mediate intercellular adhesion between fibroblasts. Fura-2 or fluo-3 labeled human fibroblasts were used to investigate calcium homeostasis during intercellular adhesion. After contact with suspended fibroblasts there was a rise in cytosolic free calcium ([Ca2+]i) and multiple calcium oscillations in substrate-attached cells. Antibodies against the extracellular but not the cytoplasmic domain of cadherin induced a similar calcium response, indicating that these responses were initiated by cadherin binding. As shown by the near-plasma membrane Ca2+ indicator (Fura-C18) and by confocal microscopy of fluo-3-loaded cells, [Ca2+]i transients probably originated at sites of cell-cell contact. Cell-cell adhesion was dependent on both calcium influx through membrane channels and release of Ca2+ from internal calcium stores, because the calcium channel inhibitor LaCl3 or pretreatment of cells with thapsigargin significantly inhibited (>35%) cell-cell attachment. The [Ca2+]i changes induced by cell-cell adhesion were temporally correlated with increased recruitment of intercellular junctional proteins into the cytoskeleton and movement of GFP-actin to sites of cell-cell contact. [Ca2+]i responses induced by intercellular adhesion were essential for both junctional protein recruitment and the establishment of strong cell-cell contacts, as loading cells with BAPTA/AM significantly inhibited cell-cell adhesion and recruitment of cadherins and beta-catenin to the actin cytoskeleton. Actin depolymerization by cytochalasin D dramatically reduced cell-cell adhesion and recruitment of cadherins and catenin to the actin cytoskeleton. These results demonstrate that cadherin-cadherin interaction induces [Ca2+]i transients during cell-cell adhesion in fibroblasts, and these calcium signals regulate cell-cell adhesion through remodeling of cortical actin and recruitment of cadherins and beta-catenin into intercellular junctions.

Use of a fluorogenic septapeptide matrix metalloproteinase assay to assess responses to periodontal treatment.

BACKGROUND: Quantification of gingival crevicular fluid matrix metalloproteinase activity may provide improved assessment of periodontal disease status and response to treatment. A fluorogenic matrix metalloproteinase substrate assay (FSA) has been developed using a methoxycoumarin-containing septapeptide analog of the alpha2(I) collagen cleavage site. This substrate exhibits increased fluorescence following cleavage by many matrix metalloproteinases, and the enzyme activity can be readily estimated with a fluorimeter. Here we compared this assay with classical methods of periodontal assessment including bleeding on probing, crevicular fluid flow, and probing depth to assess its utility as an indicator of changes in periodontal status and treatment response. METHODS: Complete measurements of probing depth were obtained for Ramfjord teeth on subjects who had been previously treated for periodontitis. Subjects were subsequently divided into groups based on existing periodontal disease severity: gingivitis (n = 21), stable periodontitis (n = 41), and severe periodontitis (n = 50). Crevicular fluid volume, bleeding on probing, and FSA were measured at each Ramfjord tooth or substitute. After baseline measurements, subjects received subgingival scaling and prophylaxis; 3 months later, they were reassessed. RESULTS: FSA measurements were positively associated with severity of disease at baseline. After treatment there were substantial reductions of FSA in gingivitis (approximately 51%; P <0.01) and severe periodontitis (approximately 45%; P <0.001), but not in stable periodontitis (13%; P >0.2). All groups showed a positive association between FSA measurements and higher bleeding scores at individual sites. FSA measurements were also positively associated with crevicular fluid flow at baseline, but after treatment there was a approximately 67% decrease (P <0.01) in the highest crevicular fluid flow class. There were significant reductions of FSA at follow-up for sites with probing depths between 0 to 3 mm (23%; P <0.05) and 4 to 6 mm (31%; P <0.05). However, the largest reduction was for sites with probing depth between 7 to 9 mm (49%; P <0.001). CONCLUSIONS: These results indicate that monitoring patients by measurement of matrix metalloproteinase levels in gingival crevicular fluid with the quenched fluorescent substrate assay provides estimates of inflammatory status, periodontal destruction, and response to treatment, especially in more severe periodontitis lesions.

Prevention of spinal cord injuries caused by diving: evaluation of the distribution and usage of a diving safety video in high schools.

OBJECTIVE: To determine and assess the distribution and use of Sudden Impact, a video designed by Think First and SportsSmart Canada, to help prevent spinal cord injury caused by careless shallow water diving among teenagers in the high risk group (15-24 years old). DESIGN: Survey of 92 public secondary schools in Toronto, Canada. SUBJECTS: The heads of the physical and health education departments of the 92 secondary public schools in the Metropolitan Toronto region. RESULTS: The response rate was 64% (59 schools), of which 76% (45) had actually received the video. Forty one schools (91%) of those that received the video reported using it. Eighty per cent of responding schools showed it to grade 11 students. Eighty per cent of schools with swimming pools used the video compared with only 42% of schools without swimming pools. CONCLUSIONS: There is a need for improvements in the system of distribution to ensure greater use of material such as this video. These may include direct distribution to principals, continuing contact with the schools, or mandatory inclusion of diving safety into the school curriculum.

Rabbit calcium-sensing receptor (CASR) gene: chromosome location and evidence for related genes.

Diverse cellular functions are regulated by the calcium-sensing receptor, encoded by the CASR gene, which plays an important role in calcium homeostasis. Here we provide the sequence for exon VII of the rabbit CASR gene and show that it is 91% identical to the human gene at the nucleotide level, and 95% identical at the amino acid level. The gene was mapped by fluorescence in situ hybridization, using a cosmid isolated from a genomic library, to chromosome 14q11 and this was confirmed independently by PCR amplification of flow sorted chromosomes. In addition, the cosmid detected sites with lower frequencies on four other chromosomes: 3q, 5p, 8p, and 13p. Two of these sites (5p and 13p) were also detected by a related but unidentical cosmid, and map to regions that are homologous to the mouse calcium-sensing receptor related sequences (Casr-rs); suggesting that they may represent CASR-related sequences in the rabbit. The data support the presence of a family of genes related to the calcium-sensing receptor in the G-protein coupled receptor (GPCR) superfamily, as well as extend the existing knowledge of homology for several human and rabbit chromosomes.

Bell's Palsy - herpes simplex virus type-1 a possible causative agent.

The role of Herpes Simplex Virus Type 1 (HSV-I) in Bell's Palsy was evaluated according to clinical symptomatology and serologic findings in a series of 41 patients seen over two years period. Four fold rise in antibody titre between the acute and convalescent sera suggesting a primary infection by HSV-1 was seen in 21.9% cases, while arise in titre or a constantly high titre suggesting a reactivation or recurrent infection by HSV-I was obtained in 29.2% cases. In 48.9% cases there was a negative antibody titre. No seasonal clustering was observed. There was no relation between the recovery of facial nerve function with the antibody titre. Key words: Bell's palsy. Herpes simplex virus.

Isolation of a variant of enterovirus 70 from a patient during an epidemic of acute haemorrhagic conjunctivitis in Pune in 1991.

We report on the isolation of enterovirus 70 (EV-70) in Pune, India in 1991. The isolate was identified by the neutralization and indirect immunofluorescent antibody tests. The isolate was found to be a prime type of the EV-70 (J670/71) which was isolated by Kono and collaborators in 1971 (Kono et al., 1974).

Interferon alfa combined with cytotoxic chemotherapy for patients with non-Hodgkin's lymphoma.

BACKGROUND: Interferon alfa has been found to be effective as an antitumor agent (with a response rate of 30 percent) in patients with low-grade non-Hodgkin's lymphoma, but its effectiveness in those with intermediate-grade non-Hodgkin's lymphoma has been less adequately tested. In a prospective randomized study we evaluated the effectiveness of adding interferon alfa to cytotoxic chemotherapy in patients with clinically aggressive, low-grade non-Hodgkin's lymphoma and certain histologic variants of intermediate-grade non-Hodgkin's lymphoma, not including diffuse histiocytic lymphoma. METHODS: The patients were randomly assigned to a regimen of cyclophosphamide, vincristine, prednisone, and doxorubicin or to this regimen combined with recombinant interferon alfa. Treatment was administered every four weeks, for 8 to 10 cycles. RESULTS: The two regimens produced comparable objective responses, but the regimen including interferon had a greater effect in prolonging the time to treatment failure (P < 0.001) and the duration of complete response (P = 0.03). Interferon alfa also had a greater effect on overall survival (P = 0.014) when the results were adjusted for important covariates. CONCLUSIONS: Interferon alfa, when added to a four-drug doxorubicin-based chemotherapy regimen, is an effective antitumor agent in patients with clinically aggressive low-grade or intermediate-grade non-Hodgkin's lymphoma.

Design considerations for the rotation of a plane premirror of a monochromator for reflecting synchrotron radiation onto the same spot of the dispersing grating of the XUV beamline.

The kinematic concept of employing a plane premirror rotated around an axis outside its surface to reflect the beam onto the same spot on the grating for three angular positions is formulated algebraically. Exact analytic solutions are found for determining the positions of the axis of rotation, the radius of the circle of rotation, and the central fixed spot on the grating in terms of the three angles and the height 2b of the grating above the incident beam. Typical numerical calculations are presented and discussed. It is generally observed that deviation of the reflected beam from the fixed spot is barely a few microns for b in centimeters.

Medical treatment and medicinal charms mentioned in the atharvanic literature.

The ancient Vedic texts Provides us with valuable information and guide lines on various multi-faced aspects of human life. The present discussion is limited to the medical treatment and medicinal charms mentioned in the Atharvanic literature with specific consideration to Kausikasutra for better understanding of the rites and actions mentioned in Atharvanaveda.