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Biomedical subjects

S K Mittal

Publications and source records attributed to S K Mittal.

At least 73 records · Page 4Linked to original sources
PubMed1995

Pathogenesis and immunogenicity of bovine adenovirus type 3 in cotton rats (Sigmodon hispidus).

Intranasal inoculation of cotton rats (Sigmodon hispidus) with 10(8) PFU of bovine adenovirus type 3 (BAd3) resulted in limited virus replication in the lung and trachea. Histopathological changes in the lungs were characterized by necrosis and hyperplasia of bronchiolar epithelium, eosinophilic intranuclear inclusions, pneumocyte type II hyperplasia in the alveoli, and mild peribronchiolar and perivascular lymphocytic infiltration. Immunohistochemically, viral antigens were observed more frequently in bronchiolar epithelial cells than in alveolar cells in cotton rat lung sections stained using a rabbit anti-BAd3 serum. Bronchiolar epithelial changes, intranuclear inclusion bodies, type II pneumocyte proliferation, peribronchiolar infiltration, and immunohistological staining were maximum at Day 3 or Day 4 postinoculation, whereas perivascular infiltration was first observed at Day 8 postinoculation. In addition to the histological study of the pathogenesis of BAd3 infection, we monitored the BAd3-specific immune response in cotton rats. Anti-BAd3 IgG and virus neutralizing antibodies were detected in sera, whereas anti-BAd3 IgA antibodies were found in the sera, lung, and nasal washes. Our results suggest that the cotton rat can serve as a useful small-animal model for investigating the pathogenesis of BAd3 infection, as well as immune responses to BAd3 recombinant virus vaccines.

Adenoviridae↗
PubMed1995

Foreign gene expression by human adenovirus type 5-based vectors studied using firefly luciferase and bacterial beta-galactosidase genes as reporters.

Adenovirus (Ad) vectors have been used extensively to obtain high-level expression of foreign genes in mammalian cells and are currently being studied for use as live viral-vectored vaccines and as gene transfer vectors for gene therapy. Many Ad recombinants have been generated that express foreign genes inserted in early region 3 (E3); however, little has been done to study the importance for gene expression of regulatory sequences flanking the gene. We have generated a series of Ad5 helper-independent vectors that contain the firefly luciferase gene or the bacterial beta-galactosidase gene (LacZ) with or without simian virus 40 (SV40) regulatory sequences, combined with E3 deletions of 1.88 or 2.69 kb. The greatest levels of luciferase expression were obtained with a vector containing the luciferase gene under the control of the SV40 promoter and polyadenylation signal inserted in a 1.88-kb E3 deletion. In contrast, LacZ expression was highest with a vector containing the LacZ gene with just the SV40 polyadenylation sequence combined with a 1.88-kb E3 deletion. It was also observed that regardless of the SV40 sequences flanking the reporter gene or the E3 deletion used, expression from the luciferase recombinants was dependent on viral DNA replication, whereas expression from the LacZ recombinants was only partially reduced when DNA replication was blocked. Analyses of RNA by dot blot hybridizations revealed that the levels of reporter gene-specific mRNA for various vectors in each series did not vary significantly. These results indicate that the kinetics and efficiency of expression of genes inserted into the E3 region, in nonconditional helper-independent vectors, may be more strongly dependent on the sequences in the foreign gene insert itself than on flanking regulatory sequences such as those used here, derived from SV40.

Adenoviruses, Human↗
PubMed1995

Development of a bovine adenovirus type 3-based expression vector.

We constructed a non-defective bovine adenovirus type 3 recombinant (BAd3-Luc) containing the firefly luciferase gene inserted in the early region 3 (E3) of the BAd3 genome. Deletion of a 696 bp XhoI-NcoI E3 segment and insertion of the luciferase gene in E3 was confirmed by Southern blot analyses. Luciferase was expressed in Madin-Darby bovine kidney cells infected with BAd3-Luc as measured by enzymic assays and Western blotting. Analyses of luciferase expression in the presence or absence of 1-beta-D-arabinofuranosylcytosine indicated that approximately 70-75% of luciferase expression was dependent on viral DNA replication, suggesting that transcription of the gene was at least partially under the control of a late promoter. Although yields of infectious virus for BAd3-Luc were approximately 10-fold lower than for wild-type virus, replication of the vector was still relatively efficient. In a Western blot experiment, anti-luciferase antibody reacted with a 62 kDa protein which is of the same molecular mass as the purified firefly luciferase polypeptide. Luciferase was also expressed in the 293 cell line infected with BAd3-Luc for at least 6 days post-infection as monitored by luciferase assays. Based on these observations we suggest that BAd-based expression vectors should have excellent potential for the development of recombinant vaccines for cattle and may also be suitable as vectors for gene transfer into human cells.

Adenoviridae↗
PubMed1995

Recombinant type 5 adenoviruses expressing bovine parainfluenza virus type 3 glycoproteins protect Sigmodon hispidus cotton rats from bovine parainfluenza virus type 3 infection.

Cotton rats were used to study the replication and pathogenesis of bovine parainfluenza virus type 3 (bPIV3) and to test the efficacy of the F and HN glycoproteins in modulating infection. In vitro cultures of cotton rat lung cells supported the growth of bPIV3 as shown by virus recovery, immunofluorescence, immunoprecipitation, and syncytium induction. Intranasal (i.n.) inoculation of cotton rats with 10(7) PFU resulted in peak recovery of virus after 2 days (8 x 10(4) PFU/g of lung tissue) and significant bronchiolitis with lymphocyte infiltration 5 to 7 days postinfection. Immunohistochemical staining of lungs and trachea demonstrated that virus antigen-positive cells increased in frequency over the course of infection to a maximum on day 5. Serum antibody responses were evaluated by enzyme-linked immunosorbent assays (ELISA), hemagglutination inhibition (HAI), and serum neutralization (SN). Following a single i.n. inoculation, serum antibody levels were 1/40,960, 1/32, and 1/80, as detected by ELISA, HAI, and SN, respectively. When an intramuscular inoculation of 10(7) PFU was administered 10 days prior to the i.n. inoculation, a secondary response which resulted in an ELISA titer of 1/163,000, an HAI titer of 1/640, and an SN titer of 1/512 was induced. IN inoculation of recombinant adenoviruses type 5 containing the bPIV3 F or HN protein or a combination of the two viruses protected cotton rats from bPIV3 challenge. Protection was evaluated serologically by ELISA, HAI, and SN titers, histopathology, immunohistochemistry, and virus recovery.

Adenoviruses, Human↗
PubMed1994

The E1 sequence of bovine adenovirus type 3 and complementation of human adenovirus type 5 E1A function in bovine cells.

The bovine adenovirus type 3 (BAV3) genome was sequenced from the left end to the HindIII site at 11%. This region comprises the entire E1 transcription unit including the open reading frames (ORF) for proteins homologous to the E1A, E1B proteins and protein IX of human adenovirus type 5 (Ad5). A portion of the BAV3 E1A protein showed significant homology with conserved region 3 (CR3), the principal transactivation region of Ad5 E1A. The BAV3 E1A protein also contains a consensus sequence known to be important for interaction with the cellular Rb protein but lacks most of the sequence corresponding to the second exon of Ad5 E1A. Promoter sequences for BAV3 E1B were not defined though the relevant region contains a 35-base pair repeat sequence. Two ORFs define the BAV3 E1B coding unit; one with regions homologous to sequences within the Ad5 E1B 19k protein, and an overlapping ORF with significant homology to the Ad5 E1B 55k protein. The encoded BAV3 E1B proteins of 157 and 420 amino acid residues (R) have predicted unmodified molecular weights of 17,393 and 46,734 respectively. Immediately following the E1B coding region there is a transcription unit containing an SP1 binding site and TATA box followed by an ORF which encodes a protein of 125R and predicted molecular weight of 13,706 with homology to protein IX of Ad5. Five concensus poly A addition sites are located in the 350 base pairs immediately following the protein IX coding region. The homology of sequences in the Ad5 E1A CR3 region and the corresponding BAV3 protein suggested that the BAV3 protein could transactivate certain Ad5 genes normally transactivated by the Ad5 E1A product. Evidence for this hypothesis was obtained in studies in which bovine cells in culture were coinfected with BAV3 and a human adenovirus type 5 (Ad5) recombinant viral vector lacking the E1A region and having a lacZ reporter gene within the E3 region dependent on E1A for its expression. Coinfection resulted in the induction of beta-galactosidase activity and the increased expression of other Ad5 early (E2A 72k) and late (hexon) proteins.

Adenoviridae↗
PubMed1994

The fibre of bovine adenovirus type 3 is very long but bent.

The sequence of the fibre of bovine adenovirus type 3 (BAd3) predicts an extremely long structure due to a large number of 15-residue repeats in the fibre shaft, the tail and head domains being similar in size to the human adenovirus fibres. The length of the fibre was confirmed using negative-strain electron microscopy of BAd3 pentons (fibre plus penton base). The fibre was found to be bent in several discrete places and the bending sites appear to correspond with irregular repeats in the shaft. We suggest that bending of the fibre is needed for the interaction of the penton base with the secondary receptors on the cell surface.

Amino Acid Sequence↗
PubMed1994

Assessment of the immune and nutritional status of the host in childhood diarrhoea due to cryptosporidium.

Cryptosporidium oocysts were detected microscopically in the concentrated faecal smears (stained by modified kinyoun's acid fast stain) in 13 out of 100 (13 per cent) cases of acute diarrhoea (AD < 2 weeks duration), 7 out of 50 (14 per cent) cases of chronic diarrhoea (CD > 2 weeks duration) and none in 50 age matched controls. The grades of malnutrition of the cases and controls were calculated by the weight for age criteria and the immune status assessed by the levels of serum immunoglobulins and SIgA in duodenal fluids. Malnutrition was observed in 6 out of 13 cases (46.1 per cent) in acute and 6 out of 7 cases (85.71 per cent) in chronic cryptosporidial diarrhoeas. There was no significant statistical difference (P > 0.05) in serum immunoglobulins and SIgA levels in chronic cryptosporidiosis. SIgA was significantly reduced (P > 0.05) in cases of acute cryptosporidiosis. Cryptosporidium is an important cause of symptomatic infection in apparently immunocompetent children not having been detected in a single non-diarrhoeal control. Further a low SIgA could contribute to acute symptomatic cryptosporidiosis by favouring colonization with the parasite.

Acute Disease↗
PubMed1993

Construction of recombinant human type 5 adenoviruses expressing rodent IL-6 genes. An approach to investigate in vivo cytokine function.

The majority of biologic functions assigned to cytokines have been characterized by in vitro assay systems which may not necessarily reflect cytokine roles in vivo. Recently, recombinant virus approaches have allowed tissue-specific expression of foreign gene products in experimental animal models. We have constructed recombinant human type 5 adenoviruses, deficient in the E3 region of the genome, with incorporated rodent IL-6 cDNA that express significant levels of biologically active IL-6 on infection both in vitro and in vivo. After i.p. injection, the liver, spleen, and peritoneum appear to be primary sites of expression, whereas the lung and bronchus are the main sites of expression after intratracheal instillation. Injection i.p. of BALB/c mice with the murine rIL-6 virus causes an increase in serum levels of bioactive IL-6 for up to 6 days post-infection, whereas similar changes are not seen in animals infected with control viruses. Coincident with enhanced plasma levels of IL-6, we detect raised serum levels of hepatic-derived acute phase proteins. Associated with the expression of IL-6 in the liver and spleen, at 7 days we note a fourfold splenomegaly with expansion of B and T cell compartments, as well as the presence of lymphoid aggregates in the liver. These morphologic changes had resolved by 16 days. Our findings demonstrate that recombinant human type 5 adenoviruses expressing cDNA for various cytokines could be used as a transient pseudo-transgenic animal model to investigate the biologic function of cytokines in vivo.

Acute-Phase Proteins↗
PubMed1993

Monitoring foreign gene expression by a human adenovirus-based vector using the firefly luciferase gene as a reporter.

We have constructed a helper-independent adenovirus type 5-luciferase recombinant (Ad5-Luc 3) containing the firefly luciferase gene flanked by simian virus 40 (SV40) regulatory sequences inserted in the early region 3 (E3) of the Ad5 genome. Expression of luciferase in cells infected with Ad5-Luc3 was relatively efficient. In HeLa cells approximately 20 micrograms luciferase per 10(6) cells was made by 36 h post-infection and a 62 kilo-Dalton (kDa) luciferase band was clearly visible in a [35S]methionine-labeled Ad5-Luc 3-infected cell extract analyzed directly by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The results of experiments in which cultured cells were infected with Ad5-Luc 3 in the presence or absence of 1-beta-D-arabinofuranosyl cytosine (AraC) showed that the majority of luciferase expression was dependent on viral DNA replication. This suggested that the enzyme was probably translated primarily from mRNA derived from transcripts expressed from the major late promoter of Ad5. An anti-luciferase antibody was raised in a rabbit and used to further characterize the luciferase expressed in HeLa cells infected with Ad5-Luc 3 by immunoprecipitations and Western blot analyses. The half-life of luciferase expressed in HeLa cells infected with Ad5-Luc 3 was calculated to be approximately 6-8 h by pulse chase analysis. Luciferase is likely to be a useful marker for monitoring virus dissemination and gene expression in experimental animals because assays for enzymatic activity are extremely sensitive and backgrounds are low in all tissues. In mice inoculated intraperitoneally (i.p.) with Ad5-Luc 3, luciferase activity was detected in the liver, spleen, kidney, and lung. A single i.p. inoculation of mice with Ad5-Luc 3 was sufficient to raise anti-luciferase antibody and Ad5 neutralizing antibody which persisted for at least 8 weeks. Even in the presence of circulating anti-luciferase and Ad5 neutralizing antibodies, luciferase activity could be detected in the livers, spleens, and kidneys of mice inoculated i.p. a second time with Ad5-Luc 3.

Adenoviridae Infections↗
PubMed1993

Magnitude of acute respiratory infections in under five.

A community-based study was carried out in a rural area of Delhi to measure the prevalence and incidence of acute respiratory infections among children below the age of 5 years. The prevalence of 12.1%, was similar in boys and girls and was seen to decline with age. The incidence of acute respiratory infections was 2.5 episodes per child per year; it was not different in boys and girls. There was a statistically significant decline in the incidence with age. Upper respiratory tract infections comprised 87.5% of total acute respiratory infection morbidity while lower respiratory tract infections were 12.5%. Both upper and lower respiratory tract infections declined with increasing age; while the former was similar among boys and girls, the incidence of latter was significantly greater in boys (0.4 episodes per year) as compared to girls (0.2 episodes per year). A total of 87.5% episodes were mild, 10.4% moderate and only 2.1% were severe. The results suggest that acute respiratory infections are a major community health problem and an acute respiratory infection control programme needs to be implemented urgently.

Acute Disease↗
PubMed1992

Sequence analysis of bovine adenovirus type 3 early region 3 and fibre protein genes.

The DNA sequences of the early region 3 (E3) and fibre protein genes of bovine adenovirus type 3 (BAd3) have been determined and the amino acid sequences predicted to be encoded by their open reading frames (ORFs) compared to those of the fibre and E3 proteins from other Ads. One of the BAd3-E3 proteins contains a region homologous to the 14.7K E3 protein of human Ad5 (HAd5). The putative BAd3 fibre protein contains a number of regions homologous to the HAd2 fibre protein sequence, but is predicted to be 244 amino acids longer owing to an increase in the number of repeating structural motifs of hydrophobic amino acid residues in the shaft region. Sequences to the left of the BAd3-E3 gene region contained the 3' end of another ORF with extensive identity with the hexon-associated protein precursor (pVIII) of HAd2. Like mouse Ad1 and canine Ad1, the BAd3 E3 gene is approximately 1.5 kb, about half the size of the E3 region of HAd2 and HAd5.

Adenoviridae↗
PubMed1992

Acute osteomyelitis: early diagnosis by ultrasonography.

A prospective study was carried out to evaluate the role of ultrasound in early detection of infections of bone. Presence of a hypoechoic collection adjacent to bone was considered highly suggestive of osteomyelitis whereas a hypoechoic collection away from the bone implied a soft tissue abscess. Cellulitis presented as increased subcutaneous thickness. Of the 31 patients clinically suspected to have osteomyelitis, and subjected to ultrasound, 25 were proven to be osteomyelitis at surgery or by subsequent radiological changes, 4 had soft tissue abscesses and the remaining 2 had cellulitis. Co-existent hip joint effusion was seen in 2 patients. It is felt that ultrasound is simple and non-invasive investigation, capable of detecting bone and soft tissue infections.

Acute Disease↗
PubMed1992

Clinical and epidemiological features of acute gastroenteritis caused by human rotavirus subgroups.

Rotavirus was detected in 44 (15.28%) of 288 hospitalised children suffering from gastroenteritis in Delhi over a period of one year. Of these 44 children, subgroup I and II rotaviruses were detected in 13 (29.55%) and 25 (56.82%) children respectively. Samples from 5 (11.36%) children did not react with either subgroup I or II monoclonal antibodies, indicating the presence of a third subgroup. Both subgroups I and II were detected in one (2.27%) case. The median ages of the children infected by subgroup I and II rotavirus were 7 and 12 months respectively. Rotavirus was more prevalent in boys than in girls. The peak of rotavirus infection was seen in the early winter months. The difference of maximum and minimum monthly temperatures correlated significantly (p less than 0.05) with the incidence of rotavirus infection. Subgroup II was the predominant group throughout the period of study and caused a significantly higher incidence of severe diarrhoea than did subgroup I.

Acute Disease↗