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P Roy

Publications and source records attributed to P Roy.

At least 19 recordsLinked to original sources
PubMed1992

Preliminary crystallographic study of bluetongue virus capsid protein, VP7.

Bluetongue virus serotype 10 (BTV-10) VP7, expressed by insect cells infected with the recombinant baculovirus, has been purified and crystallized. Two crystal forms suitable for X-ray analysis have been obtained. Type I crystals belong to space group P6(3)22 with a = b = 95.2 A, c = 181.0 A, alpha = beta = 90 degrees gamma = 120.0 degrees, and contain a single subunit in the crystallographic asymmetric unit. They diffract to dmin = 3.0 A. Type II crystals belong to space group P2(1) with a = 69.4 A, b = 97.1 A, c = 71.4 A, beta = 109.0 degrees, and contain a trimer in the crystallographic asymmetric unit. They diffract to dmin = 2.1 A. These results, together with solution studies, show that the molecule is a trimer.

Bluetongue virus↗
PubMed1992

Genomic amplification and expression of delta-endotoxin fragment of Bacillus thuringiensis.

delta-Endotoxin gene of Bacillus thuringiensis HD-1 var kurstaki codes for the insecticidal crystal protein (ICP) specific for lepidopteran insects. Since the N-terminal half of the toxin is sufficient both for insect specificity and toxicity, the coding sequence of this part of the gene CryIA(b) was amplified by PCR and cloned in pUC19. As there was no expression of immunologically detectable delta-endotoxin in this clone in E. coli, the amplified ICP gene was transferred to an expression vector pGEx2T. Restriction mapping and immunoblotting confirmed the presence and expression of the CryIA(b) gene. This insert should be suitable for expression in plant system if it is mobilized into a plant binary vector.

Bacillus thuringiensis↗
PubMed1992

Juvenile gout with typical radiographic findings.

Gout, a common disorder in adults is exceedingly rare in the first decade of life and uncommon in teenagers. A recent book on bone diseases in childhood does not even mention gout [7]. Childhood podagra with a radiographic findings is a very unusual finding indeed.

Adolescent↗
PubMed1992

Interaction of nucleic acids with core-like and subcore-like particles of bluetongue virus.

Bluetongue virus (BTV) core-like particles (CLPs) were synthesized by coexpression of VP3 and VP7 using a dual recombinant baculovirus. Purified CLPs were shown to bind single-stranded RNA in three different assay systems: gel retardation, nitrocellulose binding, and sucrose gradient sedimentation. CLPs showed equal affinity for BTV-specific and non-BTV RNA and also bound DNA. RNAase protection experiments demonstrated that bound RNA was accessible to immobilized ribonuclease, suggesting that the RNA was predominantly present on the outside of the CLPs. By using individually purified VP7 and VP3 in separate assays, the binding activity was shown to reside on VP3. These results indicate further functional homologies between BTV VP3 and the rotavirus inner-core VP2 protein.

Bluetongue virus↗
PubMed1992

Evolutionary relationships among the gnat-transmitted orbiviruses that cause African horse sickness, bluetongue, and epizootic hemorrhagic disease as evidenced by their capsid protein sequences.

The amino acid sequences of four major capsid proteins of African horse sickness virus (serotype 4, AHSV-4) have been compared with those of Bluetongue virus of sheep. Epizootic hemorrhagic disease virus of deer, and the phylogenetic relationships established. Complete nucleotide sequence analysis of three RNA segments (L2, L3, and M6) of AHSV-4 and their encoded products, VP2, VP3, and VP5, together with previously published data for VP7 (Roy et al., 1991), have revealed that of the four capsid proteins the innermost protein, VP3, is the most conserved, and the outermost protein, VP2, is the most variable. Some 57-58% of the aligned BTV-10 and EHDV-1 VP3 amino acids are identical with those of AHSV-4. This compares to an identity of 79% between the BTV and EHDV VP3 sequences. For the VP7 proteins 64% of the aligned amino acids are identical between BTV-10 and EHDV-1, while they share 44-46% amino acid residues with the aligned VP7 protein of AHSV-4. By contrast, the VP2 proteins of the three viruses share only 19-24% identical amino acids. Various other comparative analyses of the proteins indicate that the VP2 species of the three orbiviruses are similar. Unlike VP2, the other outer capsid protein, VP5 is more conserved among the three viruses. On alignment, the VP5 of AHSV-4 has some 43-45% identical amino acids with that of BTV-10 and EHDV-1. Between BTV and EHDV, 62% of the aligned sequences are identical.

African Horse Sickness Virus↗
PubMed1992

The expressed VP4 protein of bluetongue virus binds GTP and is the candidate guanylyl transferase of the virus.

A minor core protein, VP4, of bluetongue virus serotype 10 (BTV-10) has been synthesized in insect cells infected with a genetically manipulated recombinant baculovirus. When insect cells were coinfected by this recombinant virus and a recombinant baculovirus expressing the two major core proteins (VP3 and VP7) of the virus, core-like particles (CLPs) consisting of all three proteins were formed. Purified CLPs reacted with [32P]GTP which was covalently bound to VP4 only. Similarly reconstituted CLPs with VP1 or VP6 did not form covalent complexes with [32P]GTP. The virion-derived VP4 was also shown to have GTP-binding activity. The covalent binding of GTP indicates that expressed VP4 not only is biologically active but also is the candidate guanylyl transferase of the virus. The optimum reaction conditions for GTP binding by VP4 have been investigated.

Bluetongue virus↗
PubMed1992

Three-dimensional reconstruction of baculovirus expressed bluetongue virus core-like particles by cryo-electron microscopy.

When the viral proteins VP3 and VP7 of bluetongue virus (BTV) are expressed simultaneously in the baculovirus system, core-like particles form spontaneously. The 3-D structure of these core-like particles, determined from cryo-electron micrographs, reveals an icosahedral structure 72.5 nm in diameter with 200 triangular spikes arranged on a T = 13,I lattice; The five spikes around each of the fivefold axes are absent. This is in contrast to the native BTV core particles which have a complete T = 13,I lattice of 260 spikes. The spikes, attributed to VP7 trimers appear as triangular columns 8.0 nm in height with distinct inner and outer domains. The inner shell of the core-like particles, or subcore-like particle, has a T = 1 lattice composed of 60 copies of VP3. The subcore-like particle is noticeably thicker around the fivefold positions. Pores in the subcore-like particle are situated near each of the local sixfold axes, below each six-membered ring of spikes. These pores could allow the passage of metabolites and RNA to and from the core for RNA transcription during infection. It is possible that the synthetic core-like particles have an incomplete complement of VP7 spikes because the ratio of VP7 to VP3 produced in the dual expression system is less than the 13:1 required for complete core-like particles. Only the VP7 spikes which have the strongest affinity for the VP3 inner core and are involved in maintaining the structural integrity of the core-like particle are incorporated. The BTV core-like particle shows greater morphological similarity to the rotavirus than to the reovirus core particle.

Animals↗
PubMed1992

Presentation of hepatitis B virus preS2 epitope on bluetongue virus core-like particles.

A chimeric protein containing most of the hepatitis B virus preS2 region (amino acid residues 1-48) upstream to, and colinear with the amino-terminus of bluetongue virus VP7 protein (preS2-VP7) was expressed by a recombinant Autographa californica nuclear polyhedrosis virus (AcNPV). The chimeric protein formed BTV core-like particles (CLPs) in Spodoptera frugiperda cells only when the cells were coinfected with this recombinant virus and a recombinant baculovirus that expresses unmodified VP7 and VP3 of BTV. The ratio of preS2-VP7 incorporated into CLPs was influenced by the relative multiplicities of infection of the two viruses. Immunoelectron microscopy of the chimeric particles indicated that the preS2 epitope was exposed on the surface of the CLPs. When insect cells were coinfected with the preS2-VP7 recombinant virus and a baculovirus vector that synthesized only the VP3 protein, no CLPs were identified.

Amino Acid Sequence↗
PubMed1992

Protective efficacy of virus-like particles for bluetongue disease.

Bluetongue virus-like particles (VLPs) derived from multiple baculovirus expression vectors have been administered in the presence of various adjuvants to sheep, a vertebrate host susceptible to the virus, and the neutralizing antibody responses are measured. Vaccinated sheep are challenged after 4 months of inoculation, and clinical reaction indices and viraemia determined. The results indicate that these multiprotein virus-like particles lacking the genetic material are highly immunogenic and as little as 10 micrograms of VLPs in conjunction with appropriate adjuvant elicit an immune response which protects against infectious virus challenge. The formation of virus-like particles using this new technology offers a novel approach in vaccinology.

Animals↗
PubMed1992

From genes to complex structures of bluetongue virus and their efficacy as vaccines.

Bluetongue virus-like and core-like structures consisting of multiproteins in different molar ratios, have been synthesized using baculovirus multiple expression vectors. These particles lacking genetic materials, mimic the single- and double-shelled authentic virus particles and have been shown to be highly immunogenic and protective for sheep challenged with infectious virus. The formation of virus-like particles, using this new technology, offers a novel approach to vaccine development.

Animals↗
PubMed1992

Specificity of molecular hybridization techniques for the detection of bluetongue virus serotypes in Culicoides variipennis.

Direct blot hybridization (DBH) and sandwich hybridization (SH) were evaluated for their ability to detect bluetongue virus (BTV) RNA in the biting midge Culicoides variipennis (Coquillett). Probes were derived from the L3 RNA segment of BTV, serotype 17. RNA of the five BTV serotypes occurring in the USA (BTV-2, BTV-10, BTV-11, BTV-13, and BTV-17) was extracted from pools of varying numbers of infected and uninfected biting midges and assayed by direct blot and sandwich hybridization tests. Direct blot hybridization using an RNA transcript probe or cDNA probe was a fast, efficient and sensitive technique, detecting as few as one midge infected with any BTV serotype in a pool of 50 or 100. Sandwich hybridization was able to detect the homologous serotype, BTV-17, in pools containing a single infected midge in a total of 50 or 100. However, detection of the heterologous serotypes, BTV-10, BTV-11, and BTV-13, was limited to pools containing 5 or more infected midges in a total of 50, and BTV-2 was undetectable by SH. Hybridization techniques provide an alternative to the conventional detection methods of inoculation of cell culture or embryonated chicken eggs for detection of BTV.

Animals↗
PubMed1992

[Gastroesophageal reflux with combined caudal and halothane anesthesia in children].

Sixteen children, aged 2 to 5 years and ranked ASA 1, were included in this study assessing gastro-oesophageal reflux occurring under halothane anaesthesia, before and during, caudal anaesthesia. They were scheduled for surgery below the umbilicus lasting 1 to 5 h. After premedication with oral hydroxyzine (2 mg.kg-1) and intravenous atropine (10 micrograms.kg-1), induction was carried out with 3% halothane. A gastro-oesophageal pH probe was inserted via the nose after calibration at 37 degrees C. A neutral pH for the oesophageal electrode and an acid pH for the gastric one demonstrated the correct position of the probe. The pH was then registered every 4 s. The probe was left in situ until the patient left the recovery room. The caudal anaesthesia catheter was then inserted with the patient lying on his left side. Caudal anaesthesia was began with 2.5 mg.kg-1 of plain bupivacaine and 5 mg.kg-1 of plain lidocaine. When the patient was lying supine again, narcosis was maintained with 0.5% halothane and 50% nitrous oxide. A dose of 1.5 mg.kg-1 of bupivacaine was injected every 30 to 45 min. None of the children displayed any respiratory signs (coughing, dyspnoea, bronchospasm, cyanosis) during the combined anaesthetic. Two episodes of asymptomatic gastro-oesophageal reflux were revealed by this method, one lasting 7 minutes and occurring during insertion of the caudal catheter, and the other, lasting 4 minutes, during recovery. There were no pulmonary sequels. There was excellent respiratory and haemodynamic stability throughout. The two episodes seemed to have been triggered off by rapid displacement of the patient and too deep an anaesthetic.(ABSTRACT TRUNCATED AT 250 WORDS)

Anesthesia, Caudal↗
PubMed1992

Interactions between bluetongue virus core and capsid proteins translated in vitro.

To determine whether the two major core proteins (VP3 and VP7) of bluetongue virus can interact in vitro to form morphological structures, linearized VP3 and VP7 cDNA clones were transcribed using SP6 polymerase and the resultant transcripts were co-translated using rabbit reticulocyte lysates. The structures derived were isolated by sedimentation through a sucrose gradient and found to resemble VP3-VP7 core-like particles (CLPs) expressed in vivo. Reacting CLPs synthesized in vivo with outer capsid proteins translated in vitro (VP2 or VP5) indicated that each outer capsid protein has the capacity to bind to a preformed CLP. This was confirmed by in vivo expression of the appropriate genes using baculovirus vectors. The interaction of VP2 or VP5 with the CLP was analysed by electron microscopy and by using immunogold-labelled monoclonal antibody.

Antigens, Viral↗
PubMed1992

Characterization of the genes encoding two of the major capsid proteins of epizootic haemorrhagic disease virus indicates a close genetic relationship to bluetongue virus.

The sequences of the genes of two of the major capsid proteins of epizootic haemorrhagic disease virus serotype 1 (EHDV-1, Orbivirus genus, Reoviridae) have been determined by analyses of cDNA clones representing the L2 and S7 RNA segments. The EHDV-1 S7 RNA segment, which encodes the VP7 core protein, is 1162 nucleotides in length and has the capacity to encode 349 amino acids (M(r) 38,243). The EHDV-1 L2 RNA segment, which encodes the outer capsid VP2 protein (M(r) 113,249) is 2968 nucleotides in length and has an open reading frame of 971 codons. The potential secondary structure of the EHDV-1 S7 mRNA species, in particular that of the terminal regions, is comparable to those of the corresponding segments of bluetongue virus (BTV) and African horse sickness virus (AHSV); the EHDV-1 L2 mRNA species has a secondary structure similar to that of the L2 mRNA of BTV. The EHDV-1 VP2 and VP7 proteins, as well as those of the other two major structural proteins of EHDV published previously (the inner core VP3 protein and the second outer capsid, VP5), are closely related to the corresponding proteins of BTV. The EHDV and BTV VP7 sequences are more distantly related to the sequence of the AHSV VP7 protein published recently.

Amino Acid Sequence↗
PubMed1992

Expression of the major core antigen VP7 of African horsesickness virus by a recombinant baculovirus and its use as a group-specific diagnostic reagent.

The major core protein, VP7, of African horsesickness virus serotype 4 (AHSV-4), the aetiological agent of a recent outbreak of the disease in southern Europe, was expressed in insect cells infected with a recombinant baculovirus containing a cloned copy of the relevant AHSV gene (S7). Analyses of its biochemical and antigenic properties confirmed the authenticity of the protein expressed. The high-level expression of VP7 under the control of the strong polyhedrin promoter of Autographa californica nuclear polyhedrosis virus induced disc-shaped crystals in infected insect cells. This enabled us to purify the protein by a one-step ultracentrifugation procedure and to utilize it for the detection of antibodies raised in horses to various serotypes of AHSV. A serological relationship between AHSV and two other orbiviruses, bluetongue virus and epizootic haemorrhagic disease virus, was also demonstrated.

African Horse Sickness↗
PubMed1992

Multiple glycoproteins synthesized by the smallest RNA segment (S10) of bluetongue virus.

The genome of bluetongue virus, an orbivirus, consists of 10 double-stranded RNAs, each encoding at least one polypeptide. The smallest RNA segment (S10) encodes two minor nonstructural proteins, NS3 and NS3A, the structures and functions of which are not understood. We have expressed these two proteins in mammalian cells by using the T7 cytoplasmic transient expression system. Using a deletion mutant (lacking the first AUG initiation codon), we have demonstrated that the second initiation codon is used to initiate the synthesis of NS3A protein and that the two initiation codons are responsible for the synthesis not only of NS3 and NS3A but also of high-molecular-weight forms of both proteins. These higher-molecular-weight forms (GNS3 and GNS3A) are glycosylated. We have also demonstrated that the carbohydrate chains of GNS3 and GNS3A could be further modified by heterogeneous extension to polylactosaminoglycan forms. The glycosylated and nonglycosylated forms are found in similar intracellular locations in the Golgi complex. In the presence of cycloheximide, NS3 and NS3A immunofluorescence staining was pronounced in the Golgi complex, confirming that NS3 and NS3A are competent for transport to the Golgi apparatus after synthesis. We conclude that S10 gene products are integral membrane glycoproteins.

Amino Acid Sequence↗