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Biomedical subjects

J S Robertson

Publications and source records attributed to J S Robertson.

At least 37 records · Page 2Linked to original sources
PubMed1998

Direct sequencing of the HA gene of clinical equine H3N8 influenza virus and comparison with laboratory derived viruses.

Equine influenza viruses propagated in the laboratory in alternate hosts such as embryonated hens' eggs or mammalian cell culture have been analysed by HA sequencing and antigenically and their sequence compared to the original virus present in clinical material. In contrast to clinically derived human influenza virus which generally grows in MDCK cells without change, the data for equine influenza virus were less clear in that variants of equine virus were derived in both eggs and cells. The study indicated that the current use of eggs for equine influenza virus surveillance and vaccine production is entirely appropriate, but that care should be exercised when equine influenza vaccines are produced in eggs or on mammalian cell cultures.

Amino Acid Sequence↗
PubMed1997

Avian influenza A viruses differ from human viruses by recognition of sialyloligosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site.

Avian influenza virus strains representing most hemagglutinin (HA) subtypes were compared with human influenza A (H1N1,H3N2) and B virus isolates, including those with no history of passaging in embryonated hen's eggs, for their ability to bind free N-acetylneuraminic acid (Neu5Ac) and sialylollgosaccharides in a competitive binding assay and to attach to gangliosides in a solid-phase adsorption assay. The avian viruses, irrespective of their HA subtype, showed a higher affinity for sialyl-3-lactose and the other Neu5Ac2-3Gal-terminated oligosaccharides and a lower affinity for sialyl-6-lactose than for free Neu5Ac, indicative of specific interactions between the HA and the 3-linked Gal and poor accommodation of 6-linked Gal in the avian receptor-binding site (RBS). Human H1 and H3 strains, by contrast, were unable to bind to 3-linked Gal, interacting instead with the asialic portion of sialyl-6-(N-acetyllactosamine). Different parts of this moiety were recognized by H3 and H1 subtype viruses (Gal and GlcNAc, respectively). Comparison of the HA amino acid sequences revealed that residues in positions. 138, 190, 194, 225, 226, and 228 are conserved in the avian RBS, while the human HAs harbor substitutions at these positions. A characteristic feature of avian viruses was their binding to Neu5Ac2-3Gal-containing gangliosides. This property of avian precursor viruses was preserved in early human H3 isolates, but was gradually lost with further circulation of the H3 HA in humans. Consequently, later human H3 isolates, as well as H1 and type B human strains, were unable to bind to short Neu5Ac2-3Gal-terminated gangliosides, an incompatibility that correlated with higher glycosylation of the HA globular head of human viruses. Our results suggest that the RBS is highly conserved among HA subtypes of avian influenza virus, while that of human viruses displays distinctive genotypic and phenotypic variability.

Animals↗
PubMed1997

Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6'-sialyl(N-acetyllactosamine).

Synthetic sialylglycoconjugates bearing 3'-sialyllactose, 6'-sialyllactose, or 6'-sialyl(N-acetyllactosamine) moieties attached to the polyacrylic acid carrier (P-3-SL, P-6-SL, and P-6-SLN, respectively) were prepared and tested for their ability to bind to influenza virus isolates from different hosts in a competitive solid phase assay. The virus panel included egg-grown avian and porcine strains, as well as human viruses isolated and propagated solely in mammalian (MDCK) cells and their egg-adapted variants. A clear correlation was observed between the pattern of virus binding of two glycopolymers, P-3-SL and P-6-SLN, and the host species from which the virus was derived. Avian isolates displayed a high binding affinity for P-3-SL and a two to three orders of magnitude lower affinity for P-6-SLN. By contrast, all non-egg-adapted human A and B viruses bound P-6-SLN strongly but did not bind P-3-SL. Unlike the "authentic" human strains, their egg-adapted counterparts acquired an ability to bind P-3-SL, indicative of a shift in the receptor-binding phenotype toward the recognition of Neu5Ac2-3Gal-terminated sugar sequences. Among the porcine viruses and human isolates with porcine hemagglutinin, few displayed an avian-like binding phenotype, while others differed from both avian and human strains by a reduced ability to discriminate between P-3-SL and P-6-SLN. Our data show that sialylglycopolymers may become a useful tool in studies on molecular mechanisms of interspecies transfer, tissue specificity, and other structure-function relationships of the influenza virus hemagglutinin.

Amino Sugars↗
PubMed1997

Assessing the significance of reverse transcriptase activity in chick cell-derived vaccines.

A recent publication reported the detection of low levels of the enzyme reverse transcriptase (RTase) in live viral vaccines prepared in chick embryo cells. The enzyme was detected using an assay with greatly increased sensitivity compared to more conventional methods. The authors have confirmed the observation of RTase activity and demonstrate that the activity is not dependent on the production of viral vaccines in chick cells but is present ubiquitously in chick embryonic fluids. The authors have also been unable to transmit the RTase activity from chick cells to a wide variety of cells of human, monkey, rabbit and turkey origin, suggesting that the activity is not associated with an avian agent capable of infecting these cells. It is concluded that the data available present no cause for concern over the safety of vaccines derived in chick cells and current WHO requirements for such vaccines remain appropriate.

Animals↗
PubMed1996

Strategy for adventitious agent assays.

There are three important questions to be addressed when considering a strategy for adventitious virus screening. (i) What viruses should be tested for? This will depend on the species of origin of the production cell although careful consideration has to be given also to the entire manufacturing process for the possible introduction of a contaminating virus from other biological materials used in production, e.g. bovine serum. (ii) What tests should be performed? Tests for viruses are highly varied. General tests act as "catch-all" types of assays. These are complemented by a variety of tests for individual viruses or types of viruses. (iii) At what stage(s) during the manufacturing process should virus testing be performed? Manufacturing stages which should be tested for virus contamination include extensive testing of the master cell bank and limited testing of the working cell bank and end of production cells.

Animals↗
PubMed1995

A collaborative study on DNA quantitation in biological products.

In 1988, a collaborative study was set up to examine the sensitivity and reproducibility of assays for the detection of DNA in biologicals derived from continuous cell lines. Fifteen laboratories analyzed 12 samples containing different amounts of DNA and protein. When no or very little DNA was present in the test samples, false positives or overestimation was common. In contrast, when higher levels of DNA were present underestimation was the norm. The study also revealed a high degree of variability between laboratories.

Biological Products↗
PubMed1995

Replicative advantage in tissue culture of egg-adapted influenza virus over tissue-culture derived virus: implications for vaccine manufacture.

Influenza virus derived from clinical material on MDCK cells has been shown to possess haemagglutinin (HA) indistinguishable from that of the natural, uncultivated virus. In contrast, viurs derived in embryonated hens' eggs are variants with substitutions in their HA in the vicinity of the receptor binding site. We report here the superior growth of egg-adapted virus over cell-derived virus on MDCK cells in studies in which MDCK-derived virus was spiked with small amounts of egg-adapted virus and the mixture sequentially passaged on MDCK cells. Such egg-derived variants bind to and are internalized by MDCK cells with a much higher efficiency than cell-derived virus. These data imply that the natural virus, whilst able to replicate on MDCK cells, is by no means the best fit for the MDCK receptor and variants with appropriate substitutions around the receptor binding site can readily displace the natural virus. Vaccine manufacturers who are investigating the use of tissue culture for vaccine production should minimize passage levels of cell-derived virus and beware of the displacement of the original virus with variants similar to those derived in eggs, which are often antigenically distinct.

Animals↗
PubMed1995

Osteoradionecrosis of the hyoid induced by combined modality therapy for laryngeal carcinoma.

Contemporary management of laryngeal carcinoma often incorporates multiple modalities of therapy. We report a case of osteoradionecrosis of the hyoid bone in a patient treated with surgery, chemotherapy, and radiation therapy for a supraglottic squamous cell carcinoma. A discussion regarding pathophysiology, radiation dosimetry and treatment options is also presented.

Carcinoma↗
PubMed1994

Codominant mixtures of viruses in reference strains of influenza virus due to host cell variation.

Influenza viruses grown in chicken eggs may comprise mixtures of variants, creating problems in establishing international reference strains and in preparing high growth reassortants. We therefore analyzed representative reference strains of H3N2 viruses from 1987 to 1989 by direct sequencing of HA1. Three of seven reference strains had different nucleotides at the same position in nucleotide gels, indicating the presence of codominant mixtures. These nucleotide duplications occurred at residues previously shown to code for amino acids associated with egg adaptation (156, 186, and 193 of HA1). Cloning of these viruses in chicken eggs permitted separation of the mixtures, and the majority of these cloned viruses could be distinguished with monoclonal antibodies. The remaining four reference strains were homogeneous and contained one of the two amino acids usually found at these residues in HA1 (e.g., 145 Glu or Lys, 186 Ser or Ile). Analysis of epidemic H3N2 isolates, for which mammalian cell and egg isolates are available for sequence analysis from the same patient, confirmed that multiple nucleotide changes can occur at residues associated with egg adaptation. When reference or epidemic strains containing codominant mixtures were passaged in eggs one to five times, one of the codominant strains usually became dominant. Similar results were obtained with Madin Darby Canine kidney (MDCK) cells, although the dominant virus usually differed from that in eggs. Attempts to reselect an influenza virus possessing sequence changes in HA1 characteristic of mammalian cells or the original human isolate (i.e., 156 Glu, 158 Glu, 186 Ser) by multiple passages in MDCK cells were not successful, but evidence was obtained that MDCK cells can provide a selective growth advantage. Thus, variants that are dominant in eggs are not necessarily dominant in MDCK cells. To preserve the original genotype of viruses used as reference strains, we recommend the following procedure: (i) cloning in chicken eggs of the candidate virus at a very early passage, (ii) selection and analysis of multiple clones with the same ferret polyclonal and/or monoclonal antibodies used in the initial screening, and (iii) selection of the isolate whose hemagglutinin molecule most closely resembles the clinical isolate.

Animals↗
PubMed1994

Safety considerations for nucleic acid vaccines.

Whilst it is premature to formulate guidelines for the manufacturing requirements of a nucleic acid vaccine, it can never be too early to discuss and debate the issues surrounding the use of a novel biotherapeutic. The major safety issues posed by nucleic acid vaccination include the possibility of transformation (or tumorigenic) events in recipients of a DNA vaccine, the potential formation of anti-DNA antibodies, and unexpected and untoward effects of long-term expression of a foreign antigen. This paper examines the extent to which these points impinge on the safety of DNA vaccines.

Animals↗
PubMed1994

Mixed populations in influenza virus vaccine strains.

Human influenza viruses used for vaccine production have previously been adapted to grow in eggs. During egg adaptation, variants are selected and we have observed that more than one variant may derive in a single egg resulting in a mixed population. We have now investigated the extent of heterogeneity, due to host cell selection, of virus strains used for the manufacture of influenza vaccine for the 1991/1992 and 1992/1993 seasons. The A(H1N1) vaccine virus was homogeneous with respect to substitutions in the haemagglutinin deriving from egg adaptation. However, two A(H3N2) vaccine strains and the influenza B component, B/Yamagata/16/88, consisted of mixed populations, apparently due to their cultivation in eggs. The individual variants within B/Yamagata were isolated and found to be antigenically distinct. The ratios of these variants within different manufacturers' seed stocks varied to the extent that vaccine derived from them could be distinguished antigenically. Furthermore, derivation of high-growth reassortants from the A(H3N2) strains which involves passaging at limit dilution did not necessarily lead to a homogeneous virus population. The significance of these findings for the efficacy of vaccine is not known at present.

Base Sequence↗
PubMed1994

Antigenic and molecular characterization of host cell-mediated variants of equine H3N8 influenza viruses.

Antigenic differences between three of six equine influenza virus (H3N8) MDCK cell- and egg-derived pairs have been demonstrated using monoclonal and polyclonal antibodies. Sequencing of the haemagglutinin (HA) genes revealed amino acid changes in four of the six virus pairs. These data contrast with those for human isolates of influenza virus in that it was predominantly tissue culture-isolated equine virus and not egg-derived virus which displayed heterogeneity. Some of the molecular changes involved are located within the vicinity of the cell receptor-binding site (positions 156, 158 and 222) whereas others are in the vicinity of the HA1-HA2 cleavage site (positions 18 and 32 of HA1 and position 12 of HA2). Our results indicate that the host cell can play a part in selecting antigenic variants of equine influenza virus and suggest that the egg, and not cell culture as is the case for human isolates, is the preferred host for vaccine and antigenic studies.

Animals↗
PubMed1994

Polymerase chain reaction (PCR) and sequencing.

For sequence analysis of transfected genes there are two approaches which can be adopted. One is to derive a consensus sequence of the gene in question and this may, in addition to providing the basic sequence, provide limited information on the presence of variant sequences. In order to provide more extensive data on variant sequences a second approach has to be adopted and that is to subclone the transfected gene (or mRNA) and analyse the sequences of large numbers of the subclones. While there are few obstacles nowadays to deriving a consensus sequence, considerable care has to be exercised in interpreting sequence data derived after subcloning because of the possible introduction of laboratory artefacts.

Biotechnology↗