Biomedical subjects

N M McKern

Publications and source records attributed to N M McKern.

At least 19 recordsLinked to original sources

PubMed1992

Sequence data as the major criterion for potyvirus classification.

Recent knowledge of the structure of the potyvirus particle and its components appears to have resolved what was thought to be an intractable problem of plant virology. This review describes how coat-protein and gene sequence data can be used to provide an hierarchical classification of potyviruses. This classification puts the aphid and non-aphid-transmitted potyviruses into a single family, divides this family into four genera that correspond to the four modes of vector transmission, discriminates distinct potyvirus species from strains, and provides a basis for the formation of subgroups composed of closely related species within a genus.

Capsid↗

PubMed1992

Present status of the sugarcane mosaic subgroup of potyviruses.

Until recently, sugarcane mosaic virus (SCMV) was believed to be a single potyvirus consisting of a large number of strains, differing from each other in certain biological and antigenic properties. The use of affinity-purified polyclonal antibodies directed towards the surface-located, virus-specific amino termini of the coat proteins showed that 17 strains from Australia and the United States represented four distinct potyviruses, namely johnsongrass mosaic virus (JGMV), maize dwarf mosaic virus (MDMV), sorghum mosaic virus (SrMV) and SCMV. Comparisons of strains from each of these four viruses on the basis of reactions on differential sorghum and oat cultivars, cell-free translation of RNAs, morphology and serology of cytoplasmic cylindrical inclusions, amino acid sequence and peptide profiling of coat proteins, 3' non-coding nucleotide sequences, and molecular hybridization with probes corresponding to the 3' non-coding regions, resulted in exactly the same taxonomic assignments as obtained using amino-terminal serology. These results further confirm that the former sugarcane mosaic virus actually consists of four distinct viruses and show that MDMV, SrMV, and SCMV are more closely related to each other than they are to JGMV. Because these four viruses are closely related but distinct, formation of a sugarcane mosaic subgroup in the genus Potyvirus would be appropriate.

Edible Grain↗

PubMed1992

Strains of bean common mosaic virus consist of at least two distinct potyviruses.

Bean common mosaic virus (BCMV) consists of a large number of pathotypes and strains which have largely been identified by their characteristic interactions with a selected number of differential bean cultivars. The relationships among these strains and other potyviruses that infect legumes are complex, with indications that BCMV, blackeye cowpea mosaic virus (BlCMV) and azuki bean mosaic virus (AzMV) may be strains of the one virus. Using high performance liquid chromatographic peptide profiles of coat-protein digests, the NL3 and NY15 strains of BCMV were compared with each other, with the Type and W strains of BlCMV and with the mild mottle strain of peanut stripe virus (PStV). The results suggest that BCMV-NL3 and BCMV-NY15 are distinct potyviruses, not strains of the one virus, and that BCMV-NY15 is a strain of the same potyvirus that includes BlCMV, PStV, AzMV and three potyvirus isolates (74, PM, PN) from soybeans.

Capsid↗

PubMed1992

Coat protein properties suggest that azuki bean mosaic virus, blackeye cowpea mosaic virus, peanut stripe virus, and three isolates from soybean are all strains of the same potyvirus.

The interrelationship of a number of potyviruses infecting legumes has been investigated by comparing molecular properties of their coat proteins. Comparison of the coat proteins by the techniques of amino acid analysis and PAGE was inadequate to distinguish strains from distinct potyviruses. However, high-performance liquid chromatographic peptide profiles of tryptic digests of coat proteins of these legume-infecting potyviruses enabled such assignments to be made. These data indicate that amino acid sequences of coat proteins of azuki bean mosaic virus, the Type and W strains of blackeye cowpea mosaic virus, three isolates (74, PM, PN) of a potyvirus obtained from soybean in Taiwan, and the Blotch and Mild Mottle strains of peanut stripe virus (PStV) may be very similar to the known sequence of PStV Stripe coat protein. In contrast, peptide profiles of coat proteins from soybean mosaic virus, clover yellow vein virus, bean yellow mosaic virus, potato virus Y, and tobacco etch virus were dissimilar to each other and to the profile of PStV Stripe, suggesting that their coat protein sequences were also quite different. Based on observations of the coat protein structure of many potyviruses, the results suggest that the potyvirus isolates with similar coat proteins are strains of the same potyvirus.

Amino Acid Sequence↗

PubMed1991

Coat protein of potyviruses. 7. Amino acid sequence of peanut stripe virus.

The amino acid sequence of the 287-residue coat protein of peanut stripe virus (PStV) was determined from the sequences of overlapping peptide fragments. Results indicated that the amino terminus was blocked by an acetyl group, as has previously been found for the coat protein of Johnsongrass mosaic potyvirus. Comparison of the PStV sequence with coat proteins of 20 distinct potyviruses gave sequence identities of 47-57%, except for zucchini yellow mosaic virus (ZYMV), passionfruit woodiness virus (PWV), and the related strains watermelon mosaic virus 2 (WMV 2) and soybean mosaic virus-N, which showed sequence identities of 70-76%. Several amino acid residues which were common to the core sequences of these coat proteins were at positions previously found to be invariant among potyvirus coat proteins. The degree of these similarities suggests that although PStV, WMV 2, ZYMV, and PWV are distinct potyviruses, they share a common ancestor in their evolutionary development.

Amino Acid Sequence↗

PubMed1991

Physicochemical and immunological characterization of recombinant host-protective antigen (VP2) of infectious bursal disease virus.

Small fusions to the N-terminal end of the host-protective antigen (VP2) of infectious bursal disease virus lead to stable expression of VP2 in Escherichia coli and yeast, and reduce the levels of inclusion body formation in E. coli in comparison to VP2 constructs with larger N-terminal fusions. VP2 produced with small N-terminal fusions, like native viral VP2, can be fractionated into a high molecular weight 'multimeric' form and a monomeric form. A virus-neutralizing monoclonal antibody that only recognizes undenatured VP2 preferentially reacts with multimeric forms of recombinant VP2. Both native and recombinant monomeric forms of VP2 are non-immunogenic. The multimeric forms of viral and yeast-derived VP2 are highly immunogenic, while those produced in E. coli are not.

Antibodies, Monoclonal↗

PubMed1991

The effect of dissociation of Bacteroides nodosus pili on their efficacy as a protective antigen against ovine footrot.

Previous studies have shown that pili from homologous Bacteroides nodosus provide protective immunity in sheep against footrot, whereas denatured pilin subunits are ineffective. The aim of the present study was to examine whether pili that were dissociated into pilin subunits under less vigorous, non-denaturing treatment conditions, would provide an effective level of protective immunity. Using the techniques of gel permeation chromatography, light scattering and susceptibility to proteolysis as measures of disruption, it was shown that pili were dissociated either by the neutral detergents n-octyl-beta-D-glucopyranoside (NOG) and Tween 80 or by lowering the pH with 1 M phosphoric acid to pH 2.2. Circular dichroic spectra indicated, however that the samples were not denatured by these treatments. Electron microscopic monitoring of detergent dissociated material following exhaustive dialysis showed the presence of protein-detergent micelles and "in-line" aggregates which gave the appearance of short fibres. Within these monitored preparations, there was no evidence of native undissociated pili. Pili dissociated by NOG or acid were tested in protection trials and shown to provide protective immunity, although agglutination titres of serum taken from the vaccinated sheep were significantly lower than those of animals inoculated with intact pili.

Animals↗

PubMed1991

Unexpected sequence diversity in the amino-terminal ends of the coat proteins of strains of sugarcane mosaic virus.

The sequence of the 3'-terminal 1343 nucleotides of the SC strain of the sugarcane mosaic virus (SCMV-SC) genome was compared with the 1376 nucleotides at the 3' terminus of maize dwarf mosaic virus B (MDMV-B). The SCMV-SC sequence includes an open reading frame which codes for the viral coat protein of 313 amino acids (nucleotides 157 to 1116), followed by a 3' non-coding region of 235 nucleotides and a poly(A) tail. The MDMV-B sequence codes for the capsid protein (nucleotides 157 to 1139) of 328 amino acids and has a 3' non-coding region of 236 nucleotides. The coat protein of SCMV-SC has 92% identity with that of MDMV-B except for the region between amino acid residues 27 and 70 of SCMV-SC. This region of SCMV-SC is smaller (44 residues) than the equivalent region in MDMV-B (59 residues) and has only 22% identity with the MDMV-B sequence. Possible mechanisms for the generation of this sequence diversity are discussed. Despite this diversity, the sequence identities of both the major part of the coat proteins and the 3' non-coding regions confirm the proposal, based on previously described serological data, that SCMV-SC and MDMV-B are strains of SCMV.

Amino Acid Sequence↗

PubMed1990

Passive protection against infectious bursal disease virus by viral VP2 expressed in yeast.

Infectious bursal disease virus (IBDV), a pathogen of major economic importance to the world's poultry industries, causes a severe immunodepressive disease in young chickens. Maternal antibodies are able to protect the progeny passively from IBDV infection. The gene encoding the IBDV host-protective antigen (VP2) has been cloned and expressed in yeast resulting in the production of an antigen that very closely resembles native VP2. When injected into specific pathogen free chickens a single dose of microgram quantities of the yeast derived antigen induces high titres of virus neutralizing antibodies that are capable of passively protecting young chickens from infection with IBDV.

Amino Acid Sequence↗

PubMed1989

Coat protein of potyviruses. 6. Amino acid sequences suggest watermelon mosaic virus 2 and soybean mosaic virus-N are strains of the same potyvirus.

The amino acid sequence of the coat protein of watermelon mosaic virus 2 (WMV 2) was determined by a combination of peptide and nucleic acid sequencing. The coat protein of WMV 2 contained 281 amino acid residues including a single cysteine at position 132 and a blocked amino terminus. Comparison with the coat protein sequences of 20 strains of ten distinct potyviruses showed sequence homologies ranging from 43% to 69% except for the N strain of soybean mosaic virus (SMV-N), where the sequence homology with WMV 2 was 83%. This degree of homology and the location of sequence differences between WMV 2 and SMV-N is much closer to that observed between strains of the same virus than that found between distinct potyviruses. These data suggest that WMV 2 and SMV-N may be strains of the same virus.

Amino Acid Sequence↗

PubMed1989

Sequence of pilin from Bacteroides nodosus 351 (Serogroup H) and implications for serogroup classification.

The nucleotide sequence of the pilin gene from Bacteroides nodosus strain 351, currently classified as serogroup H, subgroup 2 (H2) has been determined. The gene encodes a single polypeptide (prepilin) of 160 amino acids and Mr 17,150. However, pilin isolated from B. nodosus 351 migrates as two distinct bands in sodium dodecyl sulphate-polyacrylamide gel electrophoresis, due to an internal peptide bond cleavage. Amino acid sequence studies of pilin from B. nodosus 351 have established that the cleavage occurs between 72Ala and 73Ser of the mature protein sequence. Comparisons of gene and amino acid sequences of pilin from B. nodosus 351 with the corresponding sequences from strains of serogroups D and H1 indicate that these sequences share a close relationship. However, the level of sequence identity between B. nodosus 351 pilin and pilin from strain 265 of serogroup H1 is lower than anticipated for strains within a serogroup and suggests that B. nodosus 265 and B. nodosus 351 should not be classified within the same serogroup.

Amino Acid Sequence↗

PubMed1988

Amino acid sequences of pilins from serologically distinct strains of Bacteroides nodosus.

Amino acid sequences of pilin from a strain of Bacteroides nodosus from serogroup B (234) and serogroup C (217) were determined. The amino-terminal N-methylphenlalanine residue of both proteins was followed by a hydrophobic sequence of 30 residues closely related to the N-terminal sequence of other pili having an amino-terminal residue of N-methylphenylalanine. These data lend support to the hypothesis that in pilins of this type, the amino-terminal sequence functions as a transport signal necessary for pilin to reach its external environment, as well as promoting intersubunit interactions for maintenance of the structural integrity of the pilus. Two hydrophilic hypervariable regions can be discerned across the pilin sequences, indicating possible locations of antigenic domains.

Amino Acid Sequence↗

PubMed1988

Coat protein of potyviruses. 4. Comparison of biological properties, serological relationships, and coat protein amino acid sequences of four strains of potato virus Y.

Four strains of potato virus Y, PVY-D, PVY-10, PVY-18, and PVY-43, obtained from different Australian sources were compared on the basis of their biological, serological and coat protein structural properties. Each of the strains could be distinguished on the basis of their reactions on selected test plant species. Two of the PVY strains, PVY-D and PVY-10, induced symptoms similar to those produced by the PVYO strain group. The reactions of PVY-18 and PVY-43, although comparable to PVYN in some hosts, did not completely match the description of the PVYN strain group. In contrast to the other three strains, PVY-18 could not be transmitted by Myzus persicae in repeated tests. No difference was observed in the serological properties of the four PVY strains in different assay systems, using polyclonal antisera. The amino acid sequences of the coat proteins of PVY-10, PVY-18, and PVY-43 were obtained and compared with the coat protein sequences of pepper mottle virus (PeMV) [Dougherty WG, Allison RF, Parks TD, Johnston RE, Feild MJ, Armstrong FB (1985) Virology 146: 282-292] and PVY-D [Shukla DD, Inglis AS, McKern NM, Gough KH (1986) Virology 152: 118-125]. The homology between the PVY strains ranged from 96.3 to 99.3% and with the PeMV sequence, 91.4 to 92.9%. Based on this high sequence homology, and the previous observation that coat protein sequences of potyvirus strains are always greater than 90% identical, PeMV could be considered a strain of PVY. However, PVY and PeMV are reported to be only distantly serologically related and on this basis PeMV is currently considered to be an independent member of the Potyvirus group.

Amino Acid Sequence↗

PubMed1988

Coat protein of potyviruses. 5. Symptomatology, serology, and coat protein sequences of three strains of passionfruit woodiness virus.

Three strains of passionfruit woodiness virus, Tip Blight (PWV-TB), Severe (PWV-S) and Mild (PWV-M), were compared on the basis of their biological, serological and coat protein structural properties. Each of the strains could be distinguished on the basis of their reactions on selected test plant species but no differences were observed in the serological properties of the three PWV strains. Molecular weight estimates on SDS-PAGE suggest the PWV coat protein contains 275 amino acid residues and sequence data for 269 of these residues is presented. The amino terminal peptide is blocked and has not been sequenced. The coat proteins of PWV-TB and PWV-S, which cause severe symptoms in infected plants, showed only three sequence differences compared to the eleven or twelve sequence changes between their coat proteins and that of the mild strain. The high sequence homology (96-99%) between the three PWV strains is similar to that previously reported for two strains of tobacco etch virus and three strains of sugarcane mosaic virus. Sequence homology between the three strains of PWV and published sequences for the coat proteins of eight distinct potyviruses ranged from 43-71% (average 57%).

Amino Acid Sequence↗

PubMed1986

Genomic structure of the large RNA segment of infectious bursal disease virus.

The larger RNA segment of infectious bursal disease virus (IBDV: Australian strain 002-73) has been characterized by cDNA cloning and nucleotide sequence analysis. We believe IBDV is the first birnavirus to be sequenced and so have confirmed the coding region by N-terminal amino acid sequence analysis of intact viral proteins and several tryptic peptide fragments. The large RNA segment encodes in order the 37-kDa, 28-kDa and 32-kDa proteins within a continuous open reading frame and the primary translation product appears to be subsequently processed into the mature viral proteins. The large protein precursor is still processed into the 32-kDa host protective immunogen when expressed as a fusion protein in E. coli. These results are in marked contrast to the predictions from in vitro translation data that birnavirus genomes are expressed as polycistronic templates. We can now propose that birnaviruses, in particular IBDV, possess monocistronic segments and that the precursor is proteolytically processed in vivo. The sequence data presented for the 32-kDa host protective immunogen may provide the basic information needed for the production of an effective subunit vaccine against this commercially important virus.

Amino Acid Sequence↗

PubMed1986

Predicted sequence of the host-protective immunogen of infectious bursal disease virus.

The genome of Australian strain 002-73 of infectious bursal disease virus (IBDV) has been cloned as cDNA fragments into an expression library based on pUR plasmid vectors. Recombinant colonies were selected with a monoclonal antibody specific for the 32-kDa host-protective immunogen of IBDV and fully characterized by nucleotide sequence analysis. The amino acid sequence of several tryptic peptides derived from the native 32-kDa structural protein has confirmed the coding region assignment and nucleotide sequence data. We believe this to be the first published sequence of a birnavirus-encoded protein and these data may provide the basis for an effective subunit vaccine against IBDV.

Amino Acid Sequence↗

PubMed1986

Nucleotide sequence of the gene encoding the two-subunit pilin of Bacteroides nodosus 265.

The nucleotide sequence of the gene encoding pilin from Bacteroides nodosus 265 has been determined. The pilin is encoded by a single-copy gene, from which can be predicted a prepilin comprising a single protein chain of Mr 16,637. The prepilin sequence differs in several respects from the mature protein sequence. Seven additional N-terminal amino acid residues are present in prepilin, whereas residue 8, phenylalanine, undergoes posttranslational modification to become the N-methylated amino-terminal residue of mature pilin. In addition, further processing occurs through internal cleavage to produce two noncovalently linked subunits characteristic of pilins from serogroup H of B. nodosus, of which strain 265 is a member. The position of cleavage has been identified between alanine residues at positions 72 and 73 of the mature 149-residue pilin protein. The predicted pilin sequence of B. nodosus 265 shows extensive N-terminal amino acid sequence homology with other pilins of the N-methylphenylalanine type. In addition this sequence also shows homology with these N-methylphenylalanine-type pilins in the C-terminal region of the molecule, especially with pilin from Pseudomonas aeruginosa PAK.

Amino Acid Sequence↗

PubMed1986

Expression of pili from Bacteroides nodosus in Pseudomonas aeruginosa.

The pili of Bacteroides nodosus, the causative agent of ovine footrot, constitute the major host-protective immunogen against homologous serotypic challenge. The pilin gene from B. nodosus 198 has been cloned and morphologically expressed as extracellular pili in Pseudomonas aeruginosa by using a plasmid-borne, thermoregulated expression system. B. nodosus pilin could not be detected in cultures of P. aeruginosa grown at 32 degrees C, but after induction at 37 degrees C, B. nodosus pili were expressed on the cell surface of P. aeruginosa to the virtual exclusion of the host cell pili. Pili harvested from induced P. aeruginosa cultures were used to immunize sheep against footrot. The serum agglutinating antibody titers of vaccinated sheep were comparable to those of sheep receiving pili from B. nodosus. Subsequent challenge of the sheep with B. nodosus 198 indicated that the recombinant- DNA-derived pili vaccine and the B. nodosus pili vaccine provided similar levels of protection against footrot.

Animals↗