The genome of cyprinid herpesvirus 3 encodes 40 proteins incorporated in mature virions.

Koi herpesvirus, also known as cyprinid herpesvirus 3 (CyHV-3), is the aetiological agent of an emerging and mortal disease in common and koi carp. CyHV-3 virions present the characteristic morphology of other members of the order Herpesvirales, being composed of an envelope, a capsid containing the genome and a tegument. This study identified CyHV-3 structural proteins and the corresponding encoding genes using liquid chromatography tandem mass spectrometry-based proteomic approaches. In addition, exponentially modified protein abundance index analyses were used to estimate the relative abundance of the identified proteins in CyHV-3 virions. These analyses resulted in the identification of 40 structural proteins, which were classified based on bioinformatic analyses as capsid (three), envelope (13), tegument (two) and unclassified (22) structural proteins. Finally, a search for host proteins in purified CyHV-3 virions indicated the potential incorporation of up to 18 distinct cellular proteins. The identification of the proteins incorporated into CyHV-3 virions and determination of the viral genes encoding these proteins are key milestones for further fundamental and applied research on this virus.

The major portal of entry of koi herpesvirus in Cyprinus carpio is the skin.

Koi herpesvirus (KHV), recently designated Cyprinid herpesvirus 3, is the causative agent of a lethal disease in koi and common carp. In the present study, we investigated the portal of entry of KHV in carp by using bioluminescence imaging. Taking advantage of the recent cloning of the KHV genome as a bacterial artificial chromosome (BAC), we produced a recombinant plasmid encoding a firefly luciferase (LUC) expression cassette inserted in the intergenic region between open reading frame (ORF) 136 and ORF 137. Two viral strains were then reconstituted from the modified plasmid, the FL BAC 136 LUC excised strain and the FL BAC 136 LUC TK revertant strain, including a disrupted and a wild-type thymidine kinase (TK) locus, respectively. In vitro, the two recombinant strains replicated comparably to the parental FL strain. The FL BAC 136 LUC TK revertant strain was shown in vitro to induce a bioluminescent signal allowing the detection of single positive cells as early as 24 h postinfection, while in vivo, it induced KHV infection in carp that was indistinguishable from that induced by the parental FL strain. To identify the KHV portal of entry, carp were analyzed by bioluminescence imaging at different times postinfection with the FL BAC 136 LUC TK revertant strain. These analyses demonstrated that the skin of the fish covering the fins and also the body is the major portal of entry for KHV in carp. Finally, to further demonstrate the role of the skin as the KHV portal of entry, we constructed an original system, nicknamed "U-tube," to perform percutaneous infection restricted to the posterior part of the fish. All the data obtained in the present study demonstrate that the skin, and not the gills, is the major portal of entry for KHV in carp.

Cloning of the koi herpesvirus genome as an infectious bacterial artificial chromosome demonstrates that disruption of the thymidine kinase locus induces partial attenuation in Cyprinus carpio koi.

Koi herpesvirus (KHV) is the causative agent of a lethal disease in koi and common carp. In the present study, we describe the cloning of the KHV genome as a stable and infectious bacterial artificial chromosome (BAC) clone that can be used to produce KHV recombinant strains. This goal was achieved by the insertion of a loxP-flanked BAC cassette into the thymidine kinase (TK) locus. This insertion led to a BAC plasmid that was stably maintained in bacteria and was able to regenerate virions when permissive cells were transfected with the plasmid. Reconstituted virions free of the BAC cassette but carrying a disrupted TK locus (the FL BAC-excised strain) were produced by the transfection of Cre recombinase-expressing cells with the BAC. Similarly, virions with a wild-type revertant TK sequence (the FL BAC revertant strain) were produced by the cotransfection of cells with the BAC and a DNA fragment encoding the wild-type TK sequence. Reconstituted recombinant viruses were compared to the wild-type parental virus in vitro and in vivo. The FL BAC revertant strain and the FL BAC-excised strain replicated comparably to the parental FL strain. The FL BAC revertant strain induced KHV infection in koi carp that was indistinguishable from that induced by the parental strain, while the FL BAC-excised strain exhibited a partially attenuated phenotype. Finally, the usefulness of the KHV BAC for recombination studies was demonstrated by the production of an ORF16-deleted strain by using prokaryotic recombination technology. The availability of the KHV BAC is an important advance that will allow the study of viral genes involved in KHV pathogenesis, as well as the production of attenuated recombinant candidate vaccines.

Polyvariant mutant cystic fibrosis transmembrane conductance regulator genes. The polymorphic (Tg)m locus explains the partial penetrance of the T5 polymorphism as a disease mutation.

In congenital bilateral absence of the vas deferens patients, the T5 allele at the polymorphic Tn locus in the CFTR (cystic fibrosis transmembrane conductance regulator) gene is a frequent disease mutation with incomplete penetrance. This T5 allele will result in a high proportion of CFTR transcripts that lack exon 9, whose translation products will not contribute to apical chloride channel activity. Besides the polymorphic Tn locus, more than 120 polymorphisms have been described in the CFTR gene. We hypothesized that the combination of particular alleles at several polymorphic loci might result in less functional or even insufficient CFTR protein. Analysis of three polymorphic loci with frequent alleles in the general population showed that, in addition to the known effect of the Tn locus, the quantity and quality of CFTR transcripts and/or proteins was affected by two other polymorphic loci: (TG)m and M470V. On a T7 background, the (TG)11 allele gave a 2.8-fold increase in the proportion of CFTR transcripts that lacked exon 9, and (TG)12 gave a sixfold increase, compared with the (TG)10 allele. T5 CFTR genes derived from patients were found to carry a high number of TG repeats, while T5 CFTR genes derived from healthy CF fathers harbored a low number of TG repeats. Moreover, it was found that M470 CFTR proteins matured more slowly, and that they had a 1.7-fold increased intrinsic chloride channel activity compared with V470 CFTR proteins, suggesting that the M470V locus might also play a role in the partial penetrance of T5 as a disease mutation. Such polyvariant mutant genes could explain why apparently normal CFTR genes cause disease. Moreover, they might be responsible for variation in the phenotypic expression of CFTR mutations, and be of relevance in other genetic diseases.

[Viral homologues of G-protein-coupled receptor]. / Les homologues viraux des récepteurs cellulaires couplés aux protéines G.

G-protein-coupled receptors (GPCR) are seven transmembrane proteins that convert extracellular stimuli to cell signaling.Viral genes homologous to cellular GPCR have been described in the genome of Betaherpesvirinae, Gammaherpesvirinae and Poxviridae. The goal of this review is to summarize the knowledge available on viral GPCR (vGPCR) with a special interest for their roles in the biology and the pathogenesis of the infection. This review highlights some properties of vGPCR that are not shared by their cellular homologues and stresses the diversity of their functions in the biology of the infection.

Twenty-four novel hemophilia B mutations revealed by rapid scanning of the whole factor IX gene in a French population sample.

Full scanning of the factor IX gene by means of denaturing gradient gel electrophoresis enabled us to determine the molecular defects in 48 out of 49 hemophiliacs and to evaluate the spectrum of factor IX mutations in the French population. Our results further document the high molecular heterogeneity of the disease and the efficiency of this rapid screening method for disease-causing mutations. This direct approach, which is based on computer-aided analysis of the whole coding, promoter and exon-flanking factor IX gene sequences, proved to be helpful for carrier detection and prenatal diagnosis in most hemophilia B families, including sporadic cases. Moreover, we were able to identify 24 novel molecular defects of various natures in the factor IX gene.

Frequent occurrence of the CFTR intron 8 (TG)n 5T allele in men with congenital bilateral absence of the vas deferens.

Isolated congenital bilateral absence of the vas deferens (CBAVD) is an autosomal recessive disorder which has recently been shown to be associated with cystic fibrosis (CF) mutations. As part of an effort to understanding the genetic basis of this disorder, we have analysed the entire coding sequence and all the intron/exon boundaries of the cystic fibrosis transmembrane conductance regulator (CFTR) gene from 45 azoospermic individuals with this phenotype. We were able to detect a CFTR gene defect in 86% of chromosomes from these subjects. In addition to identifying 9 novel CFTR gene mutations, we found that a surprisingly high proportion (84%) of men with CBAVD who are heterozygous for a CF mutation carry the intron 8 polypyrimidine 5T CFTR allele on one chromosome. We hypothesise that this tight and significant (p < 10(-6)) linkage reflects the very mild impact of this mutation on CFTR gene expression. Although genetic heterogeneity cannot be excluded, CBAVD patients in whom no CFTR mutation has been detected are likely to harbour additional unidentified mild mutations. These observations have implications for the genetic counselling of CBAVD patients and CF families, and couples undergoing in vitro fertilisation procedures.

CFTR gene mutations in adults with disseminated bronchiectasis.

The severity and type of clinical manifestations are variable in patients with cystic fibrosis (CF). The respiratory syndromes in these patients consist of lung infections associated with disseminated bronchiectasis (DB), asthma, and chronic obstructive pulmonary disease. To investigate the possible involvement of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in chronic pulmonary disease in adults, we studied 32 DB patients with a clinically isolated respiratory syndrome. Careful analysis of all the CFTR gene exons and their flanking regions revealed a significantly increased frequency of CFTR gene mutations in these patients. Thirteen CFTR gene mutations were identified in sixteen different alleles. Six of these mutations, which have previously been reported as CF defects, were found on nine alleles. A further four, two of which had not previously been described (D192N and 406-2 AdeltaC), are potentially disease-causing mutations. We also identified three rare substitutions (R31C, L997F, T1220I), which could be involved in mild CFTR gene disease. Four patients were compound heterozygotes, one carried two CFTR gene mutations (possibly allelic) and six were heterozygous for a mutation. These results indicate that CFTR gene mutations may play a role in bronchiectatic lung disease, possibly in a multifactorial context. These findings have implications for genetic counselling of DB patients and their families.

Is being smart everything? The influence of student achievement on teachers' perceptions.

The purpose of this project was to explore the accuracy and predictors of teachers' perceptions of their students' metacognition, self-concept, and attributional beliefs. Nineteen third grade teachers each nominated six children from their classrooms based on the children's maths abilities (high, medium, low). The children were tested on metacognitive knowledge, self-concept, and attributional beliefs. Teachers completed questionnaires estimating each child's metacognitive abilities, academic self-concept, and attributional beliefs about the reasons underlying academic success and failure. Analyses indicated that teachers' perceptions were biased by children's abilities, as higher ability children were portrayed more favourably on all variables. Regressions of teachers' perceptions on children's scores revealed that teachers were moderately accurate in their ratings of children's metacognitive abilities, but not of their attributional beliefs or self-concepts. Implications for metacognitive and motivational models of the learner are discussed.

[The cystic fibrosis gene, its product CFTR protein and its mutations]. / Le gène de la mucoviscidose, son produit la protéine CFTR et ses mutations.

Cystic fibrosis (CF) is a fatal genetic disease primarily affecting Caucasians. Its etiology is complex, but it is chiefly a disease of electrolyte transport characterized by defects in fluid secretion by several epithelia. In this review are analyzed the data obtained since the cloning of the CF gene and the characterization of its product, the CF transmembrane conductance regulator (CFTR) protein, which has been shown to act like a cAMP-regulated chloride channel. This protein is a member of a family of ATP-binding proteins that are membrane-spanning, are found in a number of prokaryotic and eucaryotic cells, and have two ATP-binding domains. Unique to this family of proteins, the CFTR possesses an additional highly charged domain (the R domain). The majority of CF chromosomes (70%) have a single Phenylalanine codon deletion at position 508 of the protein (delta F508). A large number of other rare mutations (more than 230) have also been identified. This rapid accumulation of data is essential to genetic diagnosis and will aid in understanding the structure and function of the protein.

[Cystic fibrosis: the CFTR gene, its mutations, the genetic counseling]. / La mucoviscidose: le gène CFTR, ses mutations, le conseil génétique.

Cystic fibrosis is the most frequent autosomic recessively inherited disease in the European population. The gene implicated in this disease was cloned in 1989 but the consequences of the biochemical defect in the cell have not been fully elucidated. To date, 500 mutations of this 230 kilobase gene have been identified. These molecular anomalies each have an effect on the encoded protein (CFTR) an ion channel which appears to play a role in regulator functions. Results of the gene cloning and research into the different mutations have led to the development of effective strategies for molecular diagnosis facilitating genetic counselling for families at risk and the identification of atypical forms of the disease.

Both soluble and membrane-anchored forms of Felid herpesvirus 1 glycoprotein G function as a broad-spectrum chemokine-binding protein.

Recently, glycoprotein G (gG) of several alphaherpesviruses infecting large herbivores was shown to belong to a new family of chemokine-binding proteins (vCKBPs). In the present study, the function of Felid herpesvirus 1 (FeHV-1) gG as a vCKBP was investigated and the following conclusions were reached: (i) FeHV-1 secreted gG is a high-affinity broad-spectrum vCKBP that binds CC, CXC and C chemokines; (ii) gG is the only vCKBP expressed by FeHV-1 that binds CCL3 and CXCL1; (iii) secreted gG blocks chemokine activity by preventing their interaction with high-affinity cellular receptors; (iv) the membrane-anchored form of gG expressed on the surface of infected cells is also able to bind chemokines; and (v) the vCKBP activity is conserved among different field isolates of FeHV-1. Altogether, these data demonstrate that FeHV-1 gG is a new member of the vCKBP-4 family. Moreover, this study is the first to demonstrate that gG expressed at the surface of FeHV-1-infected cells can also bind chemokines.

A rapid, efficient, and sensitive assay for simultaneous detection of multiple cystic fibrosis mutations.

We describe the use of DGGE multiplex systems for rapid analysis of 15 exons of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, in which about half of the known CF molecular defects are clustered. We have previously determined the spectrum of mutations affecting the CFTR gene in the French population using a strategy based on denaturing gradient gel electrophoresis (DGGE) of amplified gene segments. Analysis of CF patients' DNA with five DGGE multiplex systems permitted us to characterize nearly 35% of non-delta F508 CF alleles and increased the CF allele detection rate to almost 82% in this population. This simple and rapid multiplex analysis strategy, which allows a significant proportion of the most frequent CF mutations in Caucasians to be detected, will be helpful in the implementation of genetic screening programs.

Identification of eight mutations and three sequence variations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene.

To determine cystic fibrosis (CF) defects in a sample of 224 non-delta F508 CF chromosomes, we used denaturing gradient gel multiplex analysis of CF transmembrane conductance regulator gene segments, a strategy based on blind exhaustive analysis rather than a search for known mutations. This process allowed us to detect 11 novel variations comprising two nonsense mutations (Q890X and W1204X), a splice defect (405 + 4 A-->G), a frameshift (3293delA), four presumed missense mutations (S912L, H949Y, L1065P, Q1071P), and three sequence polymorphisms (R31C or 223 C/T, 3471 T/C, and T1220I or 3791 C/T). We describe these variations, together with the associated phenotype when defects on both CF chromosomes were identified.

Psoralen-modified oligonucleotide primers improve detection of mutations by denaturing gradient gel electrophoresis and provide an alternative to GC-clamping.

Denaturing gradient gel electrophoresis (DGGE), a mutation-scanning procedure separating DNA fragments differing by as little as a single base change, is widely used in studies of genomic nucleotide sequence variability. The efficiency of the technique is greatly enhanced by attaching, through polymerase chain reaction (PCR) incorporation, a long GC-tail to the test DNA sequence which, as a result, becomes analysable throughout. As synthesis of GC-rich specific PCR primers is costly and time-consuming, we attempted to clamp the DNA fragment using a psoralen derivative (ChemiClamp) that promotes photo-induced cross-linking at one end. We found that this procedure provides an attractive alternative to GC-clamp in DGGE (and temperature gradient gel electrophoresis) and should prove useful in both research and diagnostic laboratories.

Molecular characterization of cystic fibrosis: 16 novel mutations identified by analysis of the whole cystic fibrosis conductance transmembrane regulator (CFTR) coding regions and splice site junctions.

The spectrum of cystic fibrosis (CF) mutations was determined in 105 patients by using denaturing gradient gel electrophoresis to screen the entire coding regions and adjacent cystic fibrosis transmembrane conductance regulator (CFTR) gene sequences. The nucleotide substitutions detected included 16 novel mutations, 11 previously described defects, and 11 nucleotide sequence polymorphisms. Among the novel mutations, 6 were of the missense type, 4 were nonsense mutations, 4 were frameshift defects, and 2 affected mRNA splicing. The mutations involved all the CFTR domains, including the R domain. Of the 61 non-delta F508 CF chromosomes studied, mutations were found on 36 (59%), raising the proportion of CF alleles characterized in our patient cohort to 88%. Given the efficacy of the screening method used, the remaining uncharacterized mutations probably lie in DNA sequences outside the regions studied, e.g., upstream-promoter sequences, the large introns, or putative regulatory regions. Our results further document the highly heterogeneous nature of CF mutations and provide the information required for DNA-based genetic testing.

Haplotype analysis of 94 cystic fibrosis mutations with seven polymorphic CFTR DNA markers.

We have analyzed 416 normal and 467 chromosomes carrying 94 different cystic fibrosis (CF) mutations with polymorphic genetic markers J44, IVS6aGATT, IVS8CA, T854, IVS17BTA, IVS17BCA, and TUB20. The number of mutations found with each haplotype is proportional to its frequency among normal chromosomes, suggesting that there is no preferential haplotype in which mutations arise and thus excluding possible selection for specific haplotypes. While many common mutations in the worldwide CF population showed absence of haplotype variation, indicating their recent origins, some mutations were associated with more than one haplotype. The most common CF mutations, delta F508, G542X, and N1303K, showed the highest number of slippage events at microsatellites, suggesting that they are the most ancient CF mutations. Recurrence was probably the case for 9 CF mutations (R117H, H199Y, R347YH, R347P, L558S, 2184insA, 3272-26A-->G, R1162X, and 3849 + 10kbC-->T). This analysis of 94 CF mutations should facilitate mutation screening and provides useful data for studies on population genetics of CF.

Screening for cystic fibrosis in dried blood spots of newborns.

We propose a newborn cystic fibrosis (CF) screening test based on the analysis of dried blood spot DNA by a strategy involving simple or multiplex denaturing gradient gel electrophoresis (DGGE) of PCR products of CFTR gene fragments, in conjunction with the immunoreactive-trypsin (IRT) assay. From May 1988 to May 1992 we have performed a neonatal screening programme of 42,000 newborns in Brittany. We identified 450 infants with an elevated IRT level. From this cohort, to evaluate the feasibility of measuring IRT in conjunction with mutation analysis in Guthrie cards, a pilot study was initially conducted on 200 individuals with normal IRT and 150 with raised IRT levels. Furthermore, a retrospective study was performed on 189 IRT positive cards, involving mutation scanning of exons 10 and 11 of the CFTR gene, which contains a number of frequent mutations including the deletion delta F508. We show that this approach has several implications for neonatal CF screening especially in decreasing the recall rate and detecting CF carriers.

Haemoglobin J-Biskra: a new mildly unstable alpha1 gene variant with a deletion of eight residues (alpha50-57, alpha51-58 or alpha52-59) including the distal histidine.

Single point mutation, which accounts for 92% of the 700 known variants, is the most frequent genetic defect responsible for abnormal haemoglobins. Small deletions (or insertions) involving from one to five residues are also observed, but in only approximately 5% of cases. The remaining variants produce fusion or extended haemoglobins. A deletion of eight residues, which included the distal histidine and its neighbours (alpha50-57, alpha51-58 or alpha52-59), was found in Hb J-Biskra. This new alpha-chain variant was mildly unstable in vitro only and there was no haematological or biochemical evidence of haemolysis in the affected family members. 24 nucleotides were missing in a region of the alpha1 gene showing an identical sequence of eight nucleotides at both ends. Several starting points could therefore lead to the same nucleotide and aminoacid remaining sequence. This deletion is the largest up to now reported in a haemoglobin molecule which is expressed at an almost normal level in the red blood cell. Comparison of the DNA sequences near to the deleted (or inserted) regions in the various haemoglobins carrying this type of abnormality almost always revealed the presence of a sequence that was hypothesized to slow down progression of the replication fork, and of repeats that may lead to possible secondary structures favouring slipped mispairing.