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B H Robertson

Publications and source records attributed to B H Robertson.

At least 55 records · Page 3Linked to original sources
PubMed1989

Altered hepatitis A VP1 protein resulting from cell culture propagation of virus.

The published sequence of hepatitis A virus (HAV), strain HAS-15, after 20-30 cell culture passages contains an 18 nucleotide deletion (Ovchinnikov et al., 1985) within the VP1 genome region. This results in a significant amino acid difference of the VP1 protein when this strain of HAV is compared with other published HAV sequences. Comparison of the polyacrylamide gel electrophoretic migration of HAS-15 HAV and two other strains of HAV revealed that the HAS-15 VP1 molecule migrated faster than the VP1 molecule of the other two strains. Enzymatic amplification of viral RNA derived from the original stool suspension and cell culture adapted HAS-15 using the polymerase chain reaction followed by hybridization analyses with selected synthetic oligonucleotide probes revealed that the original wild type virus did not contain the deletion. These results confirm that cell culture adapted HAS-15 contains an eighteen nucleotide deletion which apparently was selected during cell culture adaptation.

Capsid↗
PubMed1988

Large scale production of hepatitis A virus in cell culture: effect of type of infection on virus yield and cell integrity.

Approaches to cell culture propagation of hepatitis A virus (HAV) have used either acute infection by passage of infected cell lysates or supernatants into uninfected cells or the passage of persistently infected cells. The findings presented here demonstrate that the growth and recovery of purified virus from foetal rhesus monkey kidney (FRhK4) cells persistently infected with HAV isolate HAS-15 decreased over a 2 to 3 month period. In contrast, high multiplicity acute infection of FRhK4 cells with purified HAS-15 HAV resulted in degeneration of the cell monolayer 2 to 3 weeks later. Large scale propagation of acutely infected cells followed by traditional picornavirus purification procedures reproducibly yielded milligram amounts of purified virus.

Animals↗
PubMed1987

Nucleic acid sequence of the VP1 region of attenuated MS-1 hepatitis A virus.

The nucleotide sequence of the VP1 region of marmoset-attenuated hepatitis A virus (HAV), MS-1, was determined by incorporative dideoxynucleotide sequencing of the RNA obtained from purified, liver-derived virus. Comparison of this nucleotide sequence to those of four previously published isolates revealed that one of the isolates, HM-175, which was obtained from Australia and passed three times in marmosets, had a 8.5-11% nucleotide variability compared to the remaining four isolates which were isolated from North American sources. This nucleotide variability does not result in amino acid differences with the exception of two of the North American isolates, which were derived from tissue culture passage. These isolates have been shown to contain regions of variability generated by nucleotide insertions and/or deletions, while the remaining three isolates, including the Australian isolate, demonstrate limited amino acid differences within the VP1 molecule.

Amino Acid Sequence↗
PubMed1986

Structure of the hepatitis A virion: peptide mapping of the capsid region.

Milligram amounts of highly purified hepatitis A virus (HAV) were obtained from persistently infected cell cultures. The HAV polypeptides were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose for detection by an enzyme-linked immunotransfer blot procedure. The HAV nucleotide-derived amino acid sequence was subjected to computer analysis to identify potential immunogenic regions within the HAV capsid polypeptides. Synthetic peptides corresponding to selected regions of each of the larger putative capsid polypeptides were coupled to keyhole limpet hemocyanin and used to immunize rabbits. Four of six anti-HAV peptide sera were strongly reactive. Antipeptide serum generated against amino acids (a.a.) 75 through 82 reacted with the 27,000-molecular-weight (MW) polypeptide; serum against a.a. 279 through 285 reacted with the 29,000-MW HAV polypeptide; and sera against a.a. 591 through 602 and 606 through 618 reacted with the 33,000-MW HAV polypeptide. These reactions enabled the identification of the gene order of the larger HAV P1 region gene products. Our data indicate the following molecular weights: HAV VP2 or 1B, 27,000; HAV VP3 or 1C, 29,000; and HAV VP1 or 1D, 33,000.

Amino Acid Sequence↗
PubMed1985

Nucleotide and amino acid sequence coding for polypeptides of foot-and-mouth disease virus type A12.

The coding region for the structural and nonstructural polypeptides of the type A12 foot-and-mouth disease virus genome has been identified by nucleotide sequencing of cloned DNA derived from the viral RNA. In addition, 704 nucleotides in the 5' untranslated region between the polycytidylic acid tract and the probable initiation codon of the first translated gene, P16-L, have been sequenced. This region has several potential initiation codons, one of which appears to be a low-frequency alternate initiation site. The coding region encompasses 6,912 nucleotides and ends in a single termination codon, UAA, located 96 nucleotides upstream from a 3'-terminal polyadenylic acid tract. Microsequencing of radiolabeled in vivo and in vitro translation products identified the genome position of the major foot-and-mouth disease virus proteins and the cleavage sites recognized by the putative viral protease and an additional protease(s), probably of cellular origin, to generate primary and functional foot-and-mouth disease virus polypeptides.

Amino Acid Sequence↗
PubMed1985

Dose-response evaluation of a genetically engineered foot-and-mouth disease virus polypeptide immunogen in cattle.

Four groups of 9 cattle each were vaccinated with 10, 50, 250, or 1,250 micrograms of foot-and-mouth disease (FMD) virus A12 VP1 fusion protein that was produced in Escherichia coli and emulsified in an oil adjuvant. The groups given the 10 and 50 micrograms of antigen were revaccinated at 15 weeks and were challenge exposed at 30 weeks; 5 of 9 and 7 of 9 cattle, respectively, were protected from FMD virus infection. The remaining 2 groups, vaccinated with 250 or 1,250 micrograms of antigen, were revaccinated at 32 weeks and were challenge exposed at 45 weeks; 8 of 9 and 9 of 9 cattle, respectively, were protected. The results indicated that the biosynthetic polypeptide FMD vaccine was effective using vaccination intervals frequently followed with conventional whole-virus vaccines.

Animals↗
PubMed1984

Location of neutralizing epitopes defined by monoclonal antibodies generated against the outer capsid polypeptide, VP1, of foot-and-mouth disease virus A12.

The epitopes of six monoclonal antibodies generated against type A12 foot-and-mouth disease virus (FMDV) VP1 or its largest cyanogen bromide fragment (13 kd) were characterized. Five of these monoclonal antibodies neutralized viral infectivity. Solid-phase and competitive antigen binding assays using virion-derived antigens or a biosynthetic VP1 polypeptide identified two distinct neutralizing epitopes. One epitope was located between amino acid residues 145-168 of VP1 and the other between amino acids 169-179. The results indicate that antibodies reacting with two distinct areas of the VP1 polypeptide are capable of neutralizing FMD virus.

Animals↗
PubMed1984

Biochemical map of polypeptides specified by foot-and-mouth disease virus.

Pulse-chase labeling of foot-and-mouth disease virus-infected bovine kidney cells revealed stable and unstable viral-specific polypeptides. To identify precursor-product relationships among these polypeptides, antisera against a number of structural and nonstructural viral-specific polypeptides were used. Cell-free translations programmed with foot-and-mouth disease virion RNA or foot-and-mouth disease virus-infected bovine kidney cell lysates, which were shown to contain almost identical polypeptides, were immunoprecipitated with the various antisera. To further establish identity, some proteins were compared by partial protease digestion. Evidence for a membrane association of the polypeptides coded for by the middle genome region is also presented. A biochemical map of the foot-and-mouth disease virus genome was established from the above information.

Animals↗
PubMed1984

Epitopes on foot-and-mouth disease virus outer capsid protein VP1 involved in neutralization and cell attachment.

Foot-and-mouth disease virus structural protein VP1 elicits neutralizing and protective antibody and is probably the viral attachment protein which interacts with cellular receptor sites on cultured cells. To study the relationships between epitopes on the molecule related to neutralization and cell attachment, we tested monoclonal antibodies prepared against type A12 virus, isolated A12 VP1, and a CNBr-generated A12 VP1 fragment for neutralization and effect on viral absorption. The antibodies selected for analysis neutralized viral infectivity with varying efficiencies. One group of antibodies caused a high degree of viral aggregation and inhibited the adsorption of virus to cells by 50 to 70%. A second group of antibodies caused little or no viral aggregation but inhibited the adsorption of virus to cells by 80 to 90%. One antibody, which is specific for the intact virion, caused little viral aggregation and had no effect on the binding of virus to specific cellular receptor sites. Thus, at least three antigenic areas on the surface of foot-and-mouth disease virus which were involved in neutralization were demonstrated. One of the antigenic sites appears to have been responsible for interaction with the cellular receptor sites on the surface of susceptible cells.

Animals↗
PubMed1983

Identification of amino acid and nucleotide sequence of the foot-and-mouth disease virus RNA polymerase.

Foot-and-mouth disease virus (FMDV) RNA polymerase was purified from the polyethylene glycol (PEG)-treated supernatant of infected cell media by a combination of ion-exchange chromatography, membrane molecular filtration, and affinity chromatography. The purified RNA polymerase which migrated as a single band of 56,000 molecular weight on a polyacrylamide gel was subjected to automated Edman degradation and the sequence of the first 30 amino acid residues established. On the basis of previous evidence, which indicated that the RNA polymerase was the most 3'-translated polypeptide, plasmids containing cDNA mapping at the 3' end of the genome were characterized by restriction enzyme analysis and nucleotide sequencing. These investigations definitively established the derived amino acid sequence by confirmation of 28 of the amino terminal residues determined by amino acid sequence analysis; the location of the FMDV RNA polymerase coding region at the extreme 3' end of the genome, 96 nucleotides from the poly(A) tail; and the N-terminal cleavage point of the RNA polymerase from its precursor P100 was found to be a glutamic acid-glycine bond.

Amino Acid Sequence↗
PubMed1983

Identification of an exposed region of the immunogenic capsid polypeptide VP1 on foot-and-mouth disease virus.

Iodination of intact foot-and-mouth disease virus results in the selective labeling of VP1, substantiating its exposed location on the virion. A comparison of tryptic peptides revealed that a single tyrosine-containing peptide was labeled with iodine on intact or protease-cleaved virus. The labeled peptide from intact and protease-cleaved virus was characterized by molecular weight sizing and sequence analysis. Carboxypeptidase digestion of intact VP1, limited trypsin-cleaved VP1, and VP1 purified from bacterially contaminated tissue cultures yielded carboxyterminal residues of leucine, valine-arginine, and serine-alanine, respectively. The correlation of these findings with previous data on the amino acid sequence derived from nucleotide sequencing of serotypes A12 and O1 of foot-and-mouth disease virus VP1 places the probable exposed antigenic region of VP1 in a serotype-variable region including residues 136 through 144.

Amino Acid Sequence↗
PubMed1982

Foot-and-mouth disease virus: immunogenicity and structure of fragments derived from capsid protein VP and of virus containing cleaved VP.

Peptide fragments were obtained from the immunogenic capsid protein VP3, ca. 24 kilodaltons (kd), of foot-and-mouth disease virus type A12 119ab by three procedures: (1) spontaneous proteolysis of in virion VP3 in tissue cultures to produce a 15 kd peptide, designated S fragment; (2) trypsin treatment of purified virus to produce a 16 kg peptide, designated T fragment; and (3) cyanogen bromide cleavage of purified VP3 to produce a 13 kd fragment. Following isolation and purification by gel electrophoresis, VP3 and each of the three fragments were immunogenic for livestock. Lyophilization appeared to impair the immunogenicity of VP3. In addition, viruses containing VP3 fragments produced either by the spontaneous- or trypsin-induced proteolysis were as immunogenic as virus with its VP3 intact. Amino acid sequencing of N-terminal regions revealed that the S fragment was homologous with the N-terminus of VP3, whereas the 13 kd fragment possessed a unique N-terminus. Thus, putative common immunogenic amino acid sequences would appear to reside within an overlap region of the 15 kd S and 13 kd fragments. Sequencing of cDNA prepared to viral genome RNA provided three kinds of information: it (1) placed the above overlap region in the second and third quarters of VP3; (2) demonstrated that the codons for the C-terminus of VP1 and N-terminus of VP3 are contiguous; and (3) supported earlier evidence that these same codons program a chain reversal where VP1 and VP3 are joined in the precursor polyprotein.

Amino Acid Sequence↗
PubMed1982

Selective dansylation of M protein within intact influenza virions.

Exposure of purified influenza virions to [14C]dansyl chloride resulted in the covalent attachment of the dansyl chromophore to the virion. Gel electrophoresis revealed that the dansyl chromophore was specifically coupled to the internal membrane (M) protein. Purification of the M protein by gel filtration followed by cyanogen bromide cleavage and peptide fractionation revealed that four of six peptide peaks contained dansyl label. Acid hydrolysis of the separated peptide peaks followed by thin-layer chromatography revealed that dansyl label was coupled to lysine residues present in these peptides. The results of these investigations have demonstrated that the M protein molecule is the major viral polypeptide labeled when intact virions are exposed to dansyl chloride.

Amino Acid Sequence↗
PubMed1981

Cloned viral protein vaccine for foot-and-mouth disease: responses in cattle and swine.

A DNA sequence coding for the immunogenic capsid protein VP3 of foot-and-mouth disease virus A12, prepared from the virion RNA, was ligated to a plasmid designed to express a chimeric protein from the Escherichia coli tryptophan promoter-operator system. When Escherichia coli transformed with this plasmid was grown in tryptophan-depleted media, approximately 17 percent of the total cellular protein was found to be an insoluble and stable chimeric protein. The purified chimeric protein competed equally on a molar basis with VP3 for specific antibodies to foot-and-mouth disease virus. When inoculated into six cattle and two swine, this protein elicited high levels of neutralizing antibody and protection against challenge with foot-and-mouth disease virus.

Amino Acid Sequence↗
PubMed1979

Structure of the membrane protein of influenza virus. I. Isolation and characterization of cyanogen bromide cleavage products.

After cleavage of the membrane (M) protein of influenza A/WSN virus by using cyanogen bromide (CNBr), six peptide peaks representing approximate molecular weights of 6,000, 4,000, 2,200, 1,600, 1,200, and 1,000 were resolved by gel filtration on BioGel P6. Analysis by thin-layer chromatography indicates that the first, second, fourth, and fifth peaks contain single-peptide components, whereas the third and sixth peaks contain more than one peptide. By using Whatman CM52 ion-exchange chromatography in 5 M urea, four peptides were resolved from the third BioGel P6 peak. The amino acid composition of each of the purified peptides has been determined, and partial sequences were obtained for several peptides. Based on finding a blocked amino terminal residue, the 6,000-dalton fragment appears to contain the amino terminus of the M protein, whereas the carboxy terminal peptide was identified as a 2,000-dalton peptide.

Amino Acid Sequence↗
PubMed1978

Structural components of mouse mammary tumor virus. II. Isolation and purification of virion polypeptides.

Mouse mammary tumor virus (MMTV) glycoproteins and nonglycosylated polypeptides were purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Primary amino groups were labeled with fluorescamine to enable visualization of MMTV polypeptides in the gels. Protein bands were sliced from the gels and eluted with 90 to 95% recovery. Eight MMTV polypeptides, including three of the major viral components as well as five minor proteins, were routinely obtained. Double diffusion assays and immunoelectrophoresis confirmed the retention of antigenicity identical to that of untreated MMTV virions. Antisera obtained from MMTV-free BALB/c mice immunized with these purified proteins reacted with the polypeptide immunogen as well as with detergent-disrupted MMTV virions from mouse milk or cell culture. Double diffusion assays using the specific mouse antisera failed to detect any cross-reactivity among the isolated polypeptides. A hemagglutination-inhibition assay demonstrated that the ability of MMTV virions to inhibit the hemagglutinating properties of influenza virus resides in the glycosylated polypeptides gp52, gp37.7, and gp33.

Antigens, Viral↗