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J G Kusters

Publications and source records attributed to J G Kusters.

102 records · Page 6Linked to original sources
PubMed1990

Mapping of viral epitopes with prokaryotic expression products.

Several systems are available for the expression of foreign gene sequences in Escherichia coli. We describe the use of prokaryotic expression products of viral gene fragments in order to identify the regions that specify the binding sites of antibodies. This approach is particularly successful if the antigenicity does not depend on the native protein, but only on the amino acid sequence, i.e., if the epitope is sequential. Combining prokaryotic expression with the use of synthetic peptides often permits a fast and accurate mapping of an epitope. The occurrence of immunodominant sequential epitopes on the surfaces of viruses seems to be a widespread phenomenon.

Antigens, Viral↗
PubMed1990

Sequence evidence for RNA recombination in field isolates of avian coronavirus infectious bronchitis virus.

Under laboratory conditions coronaviruses were shown to have a high frequency of recombination. In The Netherlands, vaccination against infectious bronchitis virus (IBV) is performed with vaccines that contain several life-attenuated virus strains. These highly effective vaccines may create ideal conditions for recombination, and could therefore be dangerous in the long term. This paper addresses the question of the frequency of recombination of avian coronavirus IBV in the field. A method was sought to detect and quantify recombination from sequence data. Nucleotide sequences of eight IBV isolates in a region of the genome suspected to contain recombination, were aligned and compared. Phylogenetic trees were constructed for different sections of this region. Differences in topology between these trees were observed, suggesting that in three out of eight strains in vivo RNA recombinant had occurred.

Amino Acid Sequence↗
PubMed1989

Analysis of an immunodominant region of infectious bronchitis virus.

We analyzed the antigenic fine-structure of an immunodominant region in the peplomer protein of infectious bronchitis virus. This region near the N-terminus of the S2 subunit is recognized by polyclonal antisera and by the majority of mAb that cross-react with denatured protein. Despite their involvement in neutralization, epitopes in this region were conserved in different serotypes. Epitopes of four mAb and two chicken antisera were localized by using prokaryotic expression of cDNA fragments, and overlapping peptides with lengths increasing from 3 to 12 residues (PEPSCAN). We found overlapping epitopes with lengths of 6, 9, 11, and more than 17 residues. The results indicate that the expression products are antigenically equivalent to denatured protein fragments. This suggests a general strategy for the localization of sequential epitopes in large proteins. We propose that the immunodominance of the N-terminal region of S2 is explained by features of the protein structure. Presumably, this region is a protruding protein segment of about 20 residues with a high local mobility, as indicated by the antigenicity of the peptides. The conservation of the sequence points to an involvement in a molecular recognition process during infection.

Amino Acid Sequence↗
PubMed1989

Phylogeny of antigenic variants of avian coronavirus IBV.

The sequences of the peplomeric S1 protein of four serologically distinct strains of the infectious bronchitis virus (IBV), an avian coronavirus, have been determined. The S1 protein is thought to contain the serotype-specific neutralization epitopes and to be the main target of antigenic variation. An alignment with sequences of three strains published previously showed that from the 545 amino acid residues only 243 have been conserved. Clustering of substitutions suggests that most serotype determinants are located within the first 300 amino acid residues of S1. A phylogenetic tree of the S1 sequences showed very variable rates of divergence. Differences in topology with a tree based on RNAse-T1 fingerprint data indicate that some of the IBV strains have arisen by genetic recombination.

Amino Acid Sequence↗
PubMed1989

Antigenicity of the peplomer protein of infectious bronchitis virus.

To study the antigenic structure of the peplomer protein of the avian coronavirus infectious bronchitis virus, fragments from the peplomer gene were generated by restriction-enzyme cleavage or by limited DNase digestion and inserted in the Escherichia coli expression plasmid pEX (Stanley and Luzio, 1984). The antigenicity of the expression products was tested using a number of polyclonal antisera and monoclonal antibodies. The polyclonal antisera recognized different sets of epitopes in the 1162-residue sequence. The N-terminal region of one of the two subunits, S2, was recognized by all polyclonal sera and by two monoclonal antibodies. This clearly immunodominant region contains at least two adjacent or overlapping epitopes, one of which has been localized within 18 residues. The epitopes found as antigenic pEX expression products do not coincide with the regions in the S1 subunit that have been found to contain hypervariable sequences. We suggest that these regions constitute conformation dependent neutralization epitopes that cannot be detected in the pEX system. The relevance of our findings for vaccine development is discussed.

Amino Acid Sequence↗
PubMed1989

The nucleotide sequence of the first two genes of the CFA/I fimbrial operon of human enterotoxigenic Escherichia coli.

An oligonucleotide probe, derived from the N-terminal amino acid sequence of the CFA/I fimbrial subunit protein, was used to identify the gene encoding this protein within a cloned DNA fragment encoding CFA/I fimbriae. The gene (cfa b) was found and sequenced. Flanking it upstream was a gene (cfa a) encoding a protein of 206 amino acids and downstream a gene (cfa c) probably encoding an 85 kDa protein was found. This genetic organisation of the CFA/I operon differs from that of other fimbrial operons in Escherichia coli. All three proteins have signal peptides. The nucleotide sequence was analysed for homology with other sequences, secondary structure, ribosomal binding sites and possible promoter sequences. A region of dyad symmetry probably involved in the regulation of translation of the cfa c gene was found at the 5' end of this gene. A region of dyad symmetry was also observed within the cfa b gene. In front of the CFA/I operon part of insertion sequence IS2 was found. This IS2 sequence was found in a number of CFA/I plasmids, obtained from strains isolated from various geographic locations. The insertion of the IS2 element in the CFA/I operon therefore probably happened rather early during evolution of CFA/I producing Escherichia coli strains.

Amino Acid Sequence↗
PubMed1987

Molecular epidemiology of infectious bronchitis virus in The Netherlands.

Twelve Dutch isolates and the M41 strain of infectious bronchitis virus (IBV), a coronavirus of chickens, were characterized by cross-neutralization and T1 finger-printing to elucidate their evolutionary relationship. The T1 fingerprinting showed that the Dutch isolates formed two clusters. The first cluster contained strains H52, H120, D387, V1259, V1385 and V1397; the estimated sequence homology is 99%. Cluster two comprised strains D207, D274, D212, D1466, D3128 and D3896, which have about 95% sequence homology. The M41 virus did not belong to either cluster. The four different serotypes which arose in the late 1970s belonged to cluster two and appeared to be different from the vaccine strains (H52 and H120) used at that time. This indicates that the strains were newly introduced and could have arisen from a common virus. On the other hand, three recently isolated field strains were genetically closely related to the vaccine strains H120 and H52 (cluster one), suggesting that these live vaccine strains themselves could have given rise to these serologically altered field isolates. The data are relevant to the development of new vaccine strategies.

Animals↗
PubMedDate not supplied

Synthesis of long cDNA from viral RNA template.

Methods to make long and reliable cDNA from viral RNA template have been optimized. The conditions of the denaturation of the viral RNA template were most critical. For synthesis of the first DNA strand, the concentration of the primer and the presence of an RNase inhibitor were important. During the synthesis of the second strand, the incubation temperature was found to have effect on the length of the transcripts. Application of our optimized conditions on coronaviral genomic RNA as template resulted in cDNA libraries with inserts in the range of 0.5-5 kb without a separate cDNA size selection. Furthermore, a convenient variant of the alcohol precipitation and the analysis of single-stranded DNA on neutral agarose gels are described.

1-Propanol↗
PubMedDate not supplied

Human liver myeloid dendritic cells maturate in vivo into effector DC with a poor allogeneic T-cell stimulatory capacity.

We hypothesized that the relatively low immunogenicity of liver grafts might be related to a special maturation program of hepatic myeloid dendritic cells (MDC), yielding relatively immature effector MDC with weak allogeneic T-cell stimulatory capacity. To investigate whether maturation of human liver-derived MDC in vivo differs from maturation of MDC at another anatomical location, we compared the immunophenotypes and allogeneic T-cell stimulatory capacity of MDC from hepatic with those from inguinal lymph nodes (LN). MDC were purified by immunomagnetic selection from hepatic LN obtained from multi-organ donors (n = 8) and from inguinal LN of kidney transplant recipients (n = 7). MDC from hepatic LN had a significantly reduced capacity to stimulate allogeneic T-cell proliferation compared to MDC from inguinal LN. However, this was not due to an immaturity, since MDC from hepatic LN had significantly higher expressions of HLA-DR, CD80, and CD86 compared to MDC from inguinal LN. Hepatic MDC maturate in vivo to a mature type of effector MDC with relatively poor allogeneic T-cell stimulatory capacity.

Dendritic Cells↗