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PubMed1995

The tempo and mode of SIV quasispecies development in vivo calls for massive viral replication and clearance.

Simian immunodeficiency virus (SIV) quasispecies development was followed in four monkeys (Macacca fascicularis) infected by intramuscular inoculation of phage lambda-SIVmac239 DNA. Rooted phylogenetic trees were reconstructed and used to interpret the data. The rate of fixation of base substitutions varied within and between animals reaching 3.3 x 10(-2) per site per year. These data suggest that the tempo of quasispecies development requires both massive viral replication and efficient clearance of SIV. Despite this, no significant difference was found between the observed and expected ratio of synonymous/nonsynonymous substitutions, suggesting that there was little or no selection of antigenic variants in the V1 and V2 hypervariable regions of envelope.

Amino Acid Sequence↗
PubMed1996

Inhibition of HIV infection by pseudopeptides blocking viral envelope glycoprotein-mediated membrane fusion and cell death.

The RP dipeptide motif is highly conserved in the third hypervariable region (V3 loop) of the extracellular envelope glycoprotein of different types of HIV isolates. In view of this, we have designed and synthesized a construction referred to as "template assembled synthetic peptide" (TASP), in which a lysine-rich short polypeptide was used as a template to covalently anchor arrays of tripeptides, such as RPR, RPK, or KPR. The pentavalent presentation, 5(RPR)-, 5(RPK)-, or 5(KPR)-TASP, molecules manifested maximum inhibitory activity on HIV infection with a 50% inhibitory concentration value of 1-5 microM, respectively. Structure and inhibitory-activity relationship studies using analogs of 5(KPR)-TASP indicated that the positively charged side chains of the K and R residues in the tripeptide molecules are critical for the optimal inhibitory activity of the pentavalent construct. Interestingly, replacement of L-amino acid residues by D-amino acids or reduction of the peptide bond between the first two amino acids of the tripeptide generated peptide-TASP analogs active at sub-microM, concentrations. The anti-HIV action of the peptide-TASP constructs is specific, since they inhibit infection of several types of CD4-expressing cells by HIV-1 Lai and HIV-2 EHO but not by the simian SIV-mac isolate. Our results suggest that these inhibitors block three post-CD4 binding functions of the HIV envelope glycoproteins, mediation of viral entry, syncytium formation, and triggering cell death by apoptosis. As the peptide-TASP derivatives with unnatural amino acid sequences in the tripeptide moiety retain full inhibitory activity, they should provide potent protease-resistant peptide inhibitors as potential therapeutic agents for treatment of AIDS patients.

Amino Acid Sequence↗
PubMed1997

Hepatitis C virus genomic variability in untreated and immunosuppressed patients.

To investigate whether immune pressure enhances the genetic diversity of the hepatitis C virus (HCV) hypervariable region 1, nucleotide sequences were compared in multiple sera, collected longitudinally, from three untreated patients and four patients undergoing liver transplantation for HCV-related cirrhosis. A minor variant became dominant in three of three patients following transplantation and persisted unchanged for months. Compared with untreated HCV carriers, transplant recipients had fewer quasispecies, fewer nucleotide changes (1.61 and 2.58/month), fewer amino acid sequence changes (0.40 and 1.94/month), as well as higher ratio of transitional to transversional mutations (2.57 and 0.98, P < 0.02) and lower replacement to silent mutations (1.33 and 8.21, P < 0.01). The two patients with the least genomic variation died of HCV graft infection. The data suggest that HCV variants which infect the graft are selected by recipient immune pressure at the time of transplant and that preferential replication in the graft is enhanced by routine immunosuppression.

Amino Acid Sequence↗
PubMed2000

Replication ability in vitro and in vivo of equine infectious anemia virus avirulent Japanese strain.

An attenuated equine infectious anemia virus (EIAV), V26, was previously prepared by 50 passages of the Japanese virulent strain V70 in primary horse macrophage culture. The horses inoculated with this V26 virus were shown to raise neutralizing antibodies against V70 without any viremia. Here, we investigated the in vitro and in vivo replication ability of V26. Comparison of the long-terminal repeat (LTR) sequences between V26 and V70 revealed a large insertion within the LTR U3 hypervariable region of V26. V26 with the mutation in the LTR showed much higher promoter activity in vitro than V70. This is consistent with the much higher replication rate of V26 in horse primary macrophage cultures compared with V70. In sharp contrast, we failed to identify the V26-specific LTR sequence by PCR, at least in sequential samples of plasma or peripheral blood mononuclear cells derived from three horses until day 62 after V26 inoculation. In contrast, antibody responses to EIAV were observed in all horses. The results suggest that the replication ability of V26 in vivo is extremely low. When one of the horses was subsequently challenged with cell-associated V70, it was found that the horse became PCR positive for EIAV. There was no LTR mutation in EIAV genome in samples periodically prepared from the V70-challenged horse. Thus it was suggested that the LTR mutation in EIAV, which occurs during serial passage in vitro, affects EIAV replication in vitro and in vivo.

Animals↗
PubMed1999

The evolution of porcine reproductive and respiratory syndrome virus: quasispecies and emergence of a virus subpopulation during infection of pigs with VR-2332.

GP5, the principal envelope glycoprotein of porcine reproductive and respiratory syndrome virus (PRRSV), contains a hypervariable region within the ectodomain which is responsible for generating diversity in field isolates. The purpose of this study was to gain insight into the possible origin of this diversity by following GP5 sequence changes in pigs exposed to PRRSV strain VR-2332 in utero. A region of the PRRS virus genome containing portions of ORF4 and ORF5 was amplified directly from tissues of infected pigs from birth to 132 days of age. We observed the emergence of a new PRRSV population, identified by a single nucleotide change in the ectodomain. The Asp to Asn change at amino acid 34 was also found as a minor component in pigs that expressed the wild-type sequence. The results from this study suggest that the variability in the ectodomain of ORF5 is the result of positive or negative selection, of which the mechanism remains to be determined.

Amino Acid Sequence↗
PubMed2000

Characterization of modified hepatitis C virus E2 proteins expressed on the cell surface.

The envelope proteins of hepatitis C virus (HCV) are the likely targets of neutralizing antibodies and their molecular and functional characterization is relevant for vaccine development. We previously showed that surface-expressed E2 is a better immunogen than intracellular E2 and, therefore, we were interested in exploring more efficient ways to present E2 protein on the cell surface. We found that E2 targeted to the cell surface by replacement of its transmembrane domain did not bring E1 to the surface although E1 could be expressed independently on the cell surface if its transmembrane domain was similarly replaced. FACS analysis suggested that E2 expressed on the cell surface acquired its native conformation more efficiently when truncated at aa 661 than when truncated at aa 715. The shorter form of truncated E2 better retained the ability to bind the second extracellular loop (EC2) of CD81, the putative HCV receptor. Interestingly, deletion of the hypervariable region 1 (HVR1) did not perceptibly alter E2 structure; cell-surface forms of E2 lacking the HVR1 remained reactive with conformation-sensitive MAbs and were able to bind recombinant EC2 of CD81.

Amino Acids↗
PubMed2002

Structural features of envelope proteins on hepatitis C virus-like particles as determined by anti-envelope monoclonal antibodies and CD81 binding.

The envelope glycoprotein E2 of hepatitis C virus (HCV) is a major component of the viral envelope. Knowledge of its topologic features and antigenic determinants in virions is crucial in understanding the viral binding sites to cellular receptor(s) and the induction of neutralizing antibodies. The lack of a robust cell culture system for virus propagation has hampered the characterization of E2 presented on the virion. Here we report the structural features of hepatitis C virus-like particles (HCV-LPs) of the 1a and 1b genotypes as determined by various mouse and human monoclonal anti-envelope antibodies. Our results show that the E2 protein of HCV-LPs reacts with human monoclonal antibodies recognizing conformational determinants. Monoclonal antibodies (mAbs) specific for the hypervariable region 1 (HVR-1) sequence reacted strongly with HCV-LPs, suggesting that the HVR-1 is exposed on the viral surface. Several mAbs recognized both HCV-LPs with equally high affinity, indicating that the corresponding epitopes [amino acids (aa) 192-217 of E1 and aa 412-423, aa 522-531, and aa 640-653 of E2] are conserved in both genotypes and exposed on the surface of the HCV-LP. The E2 and E1/E2 dimers of 1a bound strongly to the recombinant large extracellular loop (LEL) of CD81 (CD81-LEL) of human and African green monkey, while the HCV-LP of 1a bound weakly to human CD81-LEL. E1/E2 dimers and the HCV-LPs of 1b did not bind CD81-LEL, consistent with the notion that CD81 recognition by E2 is strain-specific and does not correlate with permissiveness of infection. A model of the topology and exposed antigenic determinants of the envelope proteins of HCV is proposed.

Animals↗
PubMed1998

Interrelationship between alcohol intake, hepatitis C, liver cirrhosis, and hepatocellular carcinoma.

The discovery of a cDNA clone of hepatitis C virus (HCV) genome in 1989 has resulted in numerous reports of high rates of the prevalence of HCV antibody in patients with alcoholic liver disease, in particular, alcoholic liver cirrhosis and hepatocellular carcinoma. Thus, the interaction between alcohol intake and HCV infection has become of great importance. In terms of the effect of alcohol on HCV-RNA levels, the data are controversial; in some reports, alcohol increases HCV-RNA levels, and in the other reports it does not. There are several reports suggesting the possibility of an elevated quasi-species of hypervariable region 1 in the HCV genome caused by alcohol drinking. Recent studies have documented that alcohol intake exaggerates the responsiveness of interferon therapy for chronic hepatitis C; however, its mechanism is still obscure. Several studies have suggested the promoting effect of alcohol on the development of hepatocellular carcinoma in type C liver cirrhosis, which has been similarly observed in type B chronic liver disease, whereas its mechanism is limited to speculations.

Alcohol Drinking↗
PubMed1998

Does IBV change slowly despite the capacity of the spike protein to vary greatly?

We have sequenced that part of the spike protein (S) gene which encodes the aminoterminal and most variable quarter (hypervariable region, HVR) of the S1 subunit of 28 isolates of the 793/B (also known as CR88 and 4/91) serotype of infectious bronchitis virus (IBV) and the whole of S1 for nine of them. The isolates were from France and Britain between the years 1985 (first isolation) and 1996. The maximum nucleotide and amino acid differences between the first isolate and the others were 4.1% and 7.6%, respectively, for the whole of S1 and 7.1% and 14.6%, respectively, in the HVR. Analysis within clearly recognisable subgroups suggested that even in the HVR the nucleotide mutation rate was only 0.3 to 0.6% per year. However, there was no evidence that mutations had become fixed in a progressive manner; this serotype did not appear to be evolving. Strains isolated several years apart could be more similar than those isolated in a given year. It is likely that the amino acid changes are largely at positions where amino acid differences are tolerated rather than as a consequence of immune pressure. Reasons for this conclusion are discussed.

Animals↗
PubMed1998

Quasispecies development by high frequency RNA recombination during MHV persistence.

Recent studies suggest that infectious viruses and particularly persisting viral RNAs often exist as diverse populations or "quasispecies". We have developed an approach to characterize populations of the murine coronavirus mouse hepatitis virus (MHV) generated during persistent infection which has allowed us to begin to address the role of the viral quasispecies in MHV pathogenesis. We analyzed the population of persisting viral RNAs using reverse-transcription polymerase chain reaction amplification (RT-PCR) of the S1 "hypervariable" region of the spike gene followed by differential colony hybridization to identify spike deletion variants (SDVs) from acute and persistently infected mice. Sequence analysis revealed that mice with the most severe chronic paralysis harbored the most complex quasispecies. Mapping of the SDVs to the predicted RNA secondary structure of the spike RNA revealed that an isolated stem loop structure is frequently deleted. Overall, these results are consistent with high frequency recombination at sites of RNA secondary structure contributing to expansion of the viral quasispecies and persisting viral pathogenesis.

Animals↗
PubMed1991

Stress proteins, autoimmunity, and autoimmune disease.

At birth, the immune system is biased toward recognition of microbial antigens in order to protect the host from infection. Recent data suggest that an important initial line of defense in this regard involves autologous stress proteins, especially conserved peptides of hsp60, which are presented to T cells bearing gamma delta receptors by relatively nonpolymorphic class lb molecules. Natural antibodies may represent a parallel B cell mechanism. Through an evolving process of "physiological" autoreactivity and selection by immunodominant stress proteins common to all prokaryotes, B and T cell repertoires expand during life to meet the continuing challenge of infection. Because stress proteins of bacteria are homologous with stress proteins of the host, there exists in genetically susceptible individuals a constant risk of autoimmune disease due to failure of mechanisms for self-nonself discrimination. That stress proteins actually play a role in autoimmune processes is supported by a growing body of evidence which, collectively, suggests that autoreactivity in chronic inflammatory arthritis involves, at least initially, gamma delta cells which recognize epitopes of the stress protein hsp60. Alternate mechanisms for T cell stimulation by stress proteins undoubtedly also exist, e.g., molecular mimicry of the DR beta third hypervariable region susceptibility locus for rheumatoid arthritis by a DnaJ stress protein epitope in gram-negative bacteria. While there still is confusion with respect to the most relevant stress protein epitopes, a central role for stress proteins in the etiology of arthritis appears likely. Furthermore, insight derived from the work thus far in adjuvant-induced arthritis already is stimulating analyses of related phenomena in autoimmune diseases other than those involving joints. Only limited data are available in the area of humoral autoimmunity to stress proteins. Autoantibodies to a number of stress proteins have been identified in SLE and rheumatoid arthritis, but their pathogenetic significance remains to be established. Nevertheless, the capacity of certain stress proteins to bind to multiple proteins in the nucleus and cytoplasm both physiologically and during stress or injury to cells, suggests that stress proteins may be important elements in the "immunogenic particle" concept of the origin of antinuclear and other autoantibodies. In short, this fascinating group of proteins, so mysterious only a few years ago, has impelled truly extraordinary new lines of investigation into the nature of autoimmunity and autoimmune disease.

Animals↗
PubMed1993

Isolation and characterization of wheat triticin cDNA revealing a unique lysine-rich repetitive domain.

Polyclonal antibodies were raised against a purified 22 kDa triticin polypeptide (delta) and were used to screen a wheat seed cDNA library in the Escherichia coli expression vector lambda gt11. The isolated cDNA clones were grouped into three families based on their cross-hybridization reactions in DNA dot-blot studies. Southern blots of genomic DNAs extracted from ditelocentric and nullisomic-tetrasomic lines of Chinese Spring wheat, probes with the excised cDNA inserts, indicated that one of the three families (9 clones) had triticin clones. This was finally confirmed by comparing the predicted amino acid sequences of two of these clones (lambda Tri-12, lambda Tri-25) with the published tryptic peptide sequences of triticin. The Southern blots also showed that there is at least one triticin gene located on the short arm of each of the homoeologous group 1 chromosomes (1A, 1B, 1D), although till now no triticin protein product has been identified for the chromosome 1B. The nucleotide sequence of the largest triticin cDNA clone lambda Tri-25 (1567 bp) is presented here, and its predicted amino acid sequence shows strong homology with the legumin-like proteins of oats (12S globulin), rice (glutelin) and legume seeds. A unique feature of the triticin sequence is that it contains a lysine-rich repetitive domain, inserted in the hypervariable region of the typical legumin-like genes. Northern blotting of total RNA extracted from different stages of the developing wheat seed revealed that the triticin gene expression is switched on 5-10 days after anthesis (DAA). There was a steady increase in the level of triticin mRNA until 20 DAA, after which it started decreasing. The maximum mRNA accumulation occurred between 17 and 20 DAA. These observations conform closely with the published data on triticin protein accumulation during grain development.

Amino Acid Sequence↗
PubMed1992

Phenotypic heterogeneity associated with defective apolipoprotein B-100 and occurrence of the familial hypercholesterolemia phenotype in the absence of an LDL-receptor defect within a Canadian kindred.

Of 163 individuals with a diagnosis of heterozygous familial hypercholesterolemia (FH), only one subject was found to be positive for familial defective apo B-100 (FDB). The eight-member kindred ascertained through this subject who presented with both a clinical phenotype of FH and the FDB apo B-100 (Arg3500----Gln) mutation was studied. Plasma lipid and lipoprotein profiles, apo E phenotypes, apo B gene markers at the 3' hypervariable region and LDL-receptor haplotypes (ApaLI, PvuII, NcoI), were determined, together with LDL-receptor activity on freshly isolated blood lymphocytes. The FDB mutation, present in four relatives, was associated with three different phenotypes: FH and severe hypercholesterolemia, moderate hypercholesterolemia and normolipidemia. The FH phenotype occurred in the absence of any functional LDL-receptor defect. In homozygotes for the absence of the PvuII cutting site who had the apo B mutation, LDL-cholesterol levels were low in the presence of the apo E3/2 phenotype and high in the presence of the apo E4/4 phenotype. None of the major known environmental influences accounted for the wide range of variation in LDL-cholesterol among the affected members. Further observations in the spouse and offspring of the normolipidemic FDB subject confirmed the association of apo E4, the FDB mutation and the PvuII(-/-) genotype with high cholesterol levels. It is concluded that the phenotypic expression of the FDB mutation may vary widely as a function of the genetic environment within a family.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1995

Polymorphism of the MHC class II Eb gene determines the protection against collagen-induced arthritis.

Collagen-induced arthritis (CIA) is an animal model of auto immune polyarthritis, sharing similarities with rheumatoid arthritis (RA). Paradoxally, susceptibility to mouse CIA is controlled by the H2A loci (DQ homologous) while RA is linked to HLA.DR genes (H2E homologous). We recently showed that the E beta d molecule prevents CIA development in susceptible H2q mice. We addressed the question of whether H2Eb polymorphism will influence CIA incidence as HLA.DRB1 polymorphism does in RA. In F1 mice, only H2Ebd and H2Ebs molecules showed protection. Using recombinant B10.RDD (Ebd/b) mice, we found that CIA protection was mediated by the first domain of the E beta d molecule. Using peptides covering the third hypervariable region of the E beta chain, we found a perfect correlation between presentation of E beta peptides by the H2Aq molecule and protection on CIA. Therefore, the mechanism by which H2Eb protects against CIA seems to rely on the affinity of E beta peptides for the H2Aq molecule.

Amino Acid Sequence↗
PubMed1994

Exchanges of short polymorphic DNA segments predating speciation in feline major histocompatibility complex class I genes.

Sequence comparisons of 14 distinct MHC class I cDNA clones isolated from species representing the three major taxonomic lineages of Felidae (domestic cat lineage, ocelot lineage, and pantherine lineage) revealed that feline MHC class I alleles have highly mosaic structures with short polymorphic sequence motifs that are rearranged between alleles of individual MHC loci, between MHC class I genes within cat species, and between homologous MHC loci in different species. The pattern of sequence variation in felids supports the role of the following factors in production and maintenance of MHC variation: (1) gradual spontaneous mutation; (2) selective pressure to conserve certain residues but also to vary in hypervariable regions, notably residues that functionally participate in antigen recognition and presentation; and (3) recombination-mediated gene exchange between alleles and between related genes. The overall amount of genetic variation observed among MHC class I genes in the Felidae family is no greater than the amount of variation within any outbred cat species (i.e., domestic cat, ocelot). The occurrence of equivalent levels of polymorphism plus the simultaneous persistence of the same sequence motifs in divergent feline species suggest that most MHC class I nucleotide site polymorphism predated species divergences. Ancient polymorphisms have been transmitted through the speciation events and modern feline MHC class I alleles were derived by recombinational exchange of polymorphic sequence motifs. Moreover, some of these sequence motifs were found in other mammalian MHC class I genes, such as classical human HLA-B5, nonclassical human HLA-E class I genes, and bovine class I genes. These results raise the prospect of an ancient origin for some motifs, although the possibility of convergence in parallel mammalian radiations cannot be excluded.

Amino Acid Sequence↗
PubMed1993

HLA-DPB1 alleles in a population from south China.

Using the polymerase chain reaction (PCR) and hybridization with oligonucleotide probes, we analyzed the distribution of DPB1 alleles in 99 healthy unrelated individuals from the city of Guangzhou (Canton), South China. Twelve different DPB1 alleles were found in this panel. The most common allele was DPB1*0501 (62.6%). Other major alleles detected included DPB1*02 (DPB1*0201 and DPB1*0202), DPB1*1301, DPB1*0401, and a recently described allele, designated DPB1*2101. The hybridization pattern of DPB1*2101 showed that this allele shared sequences with DPB1*0301 and DPB1*0601 in the A and F hypervariable regions, while the C, D, and E regions were identical to those of DPB1*0202. DPB1*2101 was observed in 11% of the subjects tested. It was found to be in strong linkage disequilibrium with DRB1*1202. In family studies, segregation of the haplotype DRB1*1202, DRB3*0301, DQA1*0601, DQB1*0301, DPB1*2101 was observed. The second exon of DPB1*2101 was sequenced from codon 8 to codon 90 and the sequence, inferred from the pattern of hybridization, was confirmed. DPB1*0301, DPB1*0402, DPB1*0101, DPB1*1401, DPB1*1901, and another recently recognized allele, now designated DPB1*2401, were detected with low frequencies. DPB1*2401 had the same hybridization pattern as DPB1*0501 except for a probe that matches codons 85-90. In this region, DPB1*2401 encoded the amino acid sequence GPMTLQ instead of EAVTLQ as in DPB1*0501.

Alleles↗
PubMed1993

Polymorphisms of the apolipoprotein B and E genes and their relationship to plasma lipid variables in healthy Chinese men.

In this study we have analysed the apolipoprotein (Apo) E polymorphism and polymorphisms of the ApoB gene, including the ApoB/XbaI and ApoB/4311 diallelic polymorphisms and a hypervariable region (HVR) situated in the 3' region of the gene (ApoB/3'HVR), in a sample of healthy male subjects from Taiyuan (northern People's Republic of China). In comparison to Caucasian populations, in the Chinese sample, the XbaI2 allele (presence of cutting site; frequency 6.1%; and 95% confidence interval, 3.3-8.9) and the long HVR alleles (9.4%; 6.0-12.8) were rare, whereas the ApoB/4311 (Ser) allele (70.8%; 65.4-76.2) and the 34-repeat allele of the HVR (HVR34; 62.4%; 56.8-68.0) were frequent. In subjects having none, one, or two HVR34 alleles, the mean levels of plasma triglycerides were 2.32 +/- 1.44 (SD), 1.45 +/- 0.74, and 1.75 +/- 1.07 g/l, respectively (P < 0.007). Similar trends were observed for very low density lipoprotein (VLDL) cholesterol, LpE:B, and LpCIII:B. The frequencies of the ApoE alleles were similar to those reported in other populations of Asian origin; E2 (7.4%; 4.2-10.6), E3 (84.4%; 80.2-88.6), and E4 (8.2%; 5.0-11.4). Individuals carrying the E2 allele had a lower mean level of ApoB than E33 individuals: 0.87 +/- 0.16 and 1.00 +/- 0.22 g/l, respectively (P < 0.007). Individuals carrying the E4 allele had higher levels of ApoE than E33 individuals: 0.140 +/- 0.084 and 0.094 +/- 0.052 g/l, respectively (P < 0.004); similar trends were observed for VLDL cholesterol, triglycerides, LpE:B, and LpCIII:B. The ApoB/HVR34 and ApoE/E4 polymorphisms accounted for 10% to 15% of the variability of the plasma levels of VLDL cholesterol, ApoE, triglycerides, LpE:B, and LpCIII:B. Several lipid variables appeared to be favourably affected by specific forms of ApoB and ApoE that are particularly frequent in this Chinese population.

Adult↗
PubMed1991

Ten HLA-DR4 alleles defined by sequence polymorphisms within the DRB1 first domain.

We have studied DRB1 sequence polymorphisms associated with DR4 subtypes using DR4-specific DNA amplification and sequence-specific oligonucleotide probe (SSOP) hybridization. The 5' amplification primer was designed to hybridize with a unique sequence in the first hypervariable region (HVR) of the DRB1 second exon of all known DR4 alleles; the 3' primer was designed to hybridize with an intron sequence common to all DRB1 The specificity of the amplification step was demonstrated by double-blind testing of 105 selected DNA samples. Prospective SSOP typing of DR4 alleles was performed in 104 unrelated individuals known to be DR4-positive, including 13 who were DR4-homozygous. A DRB1 subtype corresponding with the previously defined DR4-associated specificities Dw4, Dw10, Dw13.1, Dw13.2, Dw14.1, Dw14.2, Dw15, and DwKT2 could be assigned for each of the 117 DR4 haplotypes tested. In most cases, DR4-homozygous, DRB1-heterozygous individuals could be genotyped with the panel of probes. In the course of our analysis, we identified two new DR4-related alleles, DRB1*04.CB (DRB1*0410)1 and DRB1*04.EC (DRB1*, 0411)2 which were recognized by their novel hybridization patterns. The DRB1 second exon sequence of DRB1*04.CB is identical to DRB1*0405 except at codon 86 where GTG encodes valine instead of GGT encoding glycine. DRB1*04.EC is identical to DRB1*04.CB except at codon 74 where GAG encodes glutamic acid instead of GCG encoding alanine. Our results provide further evidence that SSOP hybridization is the most effective approach available for subtyping DR4 haplotypes and identifying unrecognized variants. A similar approach should be equally informative for subtyping other DR alleles.

Alleles↗