Construction of a viable BHV1 mutant lacking mostof the short unique region

Summary.  The 8.2 kb HindIII K-fragment of bovine herpesvirus 1 (BHV1) lies entirely within the short unique region of the genome and contains all or parts of 7 coding regions. We have probed this fragment for the presence of nonessential genes by a simple, rapid method of insertional mutagenesis. Our analysis indicates that all the genes present in the K-fragment, except the glycoprotein D gene, are nonessential for replication of BHV1 in tissue culture. After inserting a copy of the glycoprotein D gene into the thymidine kinase locus of BHV1 it was possible to delete the entire HindIII K-fragment and the contiguous 0.36 kb O-fragment in one step. The deletion mutant, which contains 7 kb less BHV1 DNA than wt virus and lacks ORF1, US2, the genes for protein kinase, glycoprotein G, glycoprotein I, and most of glycoprotien E was still replication-competent. Accepted July 30, 1997 Received June 17, 1997     

Epitopes on glycoprotein E and on the glycoprotein E/glycoprotein I complex of bovine herpesvirus 1 are expressed by all of 222 isolates and 11 vaccine strains

Summary.  Glycoprotein E (gE) of bovine herpesvirus 1 (BHV1) forms a complex with glycoprotein I (gI) and plays an important role in cell-to-cell spread mechanisms of the virus, but is not essential for propagation of the virus. To study the antigenic variability of BHV1 glycoprotein E, a set of six well characterised monoclonal antibodies (MAbs) was established using BHV1 gE and gI deletion mutants, eukaryotically expressed gE and gI and pepscan analysis. Two of these MAbs reacted with a linear gE epitope (MAbs 3 and 52), two reacted with a more conformation dependent gE epitope (MAbs 61 and 81) and two reacted with epitopes formed by a complex formed between gE and glycoprotein I (MAbs 67 and 75). With these six MAbs the gE expression of 222 BHV1 isolates and 11 BHV1 modified-live vaccine strains was studied in vitro, using an immunoperoxidase monolayer assay. All 222 BHV1 isolates and 11 vaccine strains were found to react with MAbs 61, 81 and 75. Three of the 222 isolates failed to react with MAb 67 and two of the vaccines reacted very weakly with MAbs 3 and 52. Analysis of the gE genes of these five aberrant isolates and the gE glycoproteins they expressed, did not show obvious size differences compared to wild-type BHV1. We conclude that the tested gE epitopes are highly conserved, including the epitopes formed by the gI/gE complex. Received September 15, 1999/Accepted December 16, 1999     

Latency and reactivation of a thymidine kinase-negative bovine herpesvirus 1 deletion mutant

Summary A bovine herpesvirus 1 (BHV 1) mutant variant with a deletion in the thymidine kinase (TK) gene was assessed for its ability to establish latency and be reactivated in cattle. After treatment with dexamethasone, reactivated TK^– BHV 1 was isolated from one of four cattle that received virus by intravenous inoculation only, and from four of four cattle that received virus by intranasal, intravaginal, and intravenous inoculation. Results prove that TK^– BHV 1 will establish latency and can be reactivated in the natural host.     

Characterization of changes in the short unique segment of pseudorabies virus BUK-TK900 (Suivac A) vaccine strain

Summary. Mutant strains of pseudorabies virus (PRV) of reduced virulence, such as Bartha or BUK-TK900, have been used for vaccination purposes for many years. In contrast to the Bartha strain, BUK-TK900 has not been well characterised at the molecular level. The detailed analysis of this vaccine strain was urged by the fact of the isolation in Poland of field strains which were suspected to originate from BUK-TK900.We characterised changes in the U_S region of this strain, focusing our attention on gE and gI genes. The only deletion, about 300bp, found in BamHI 7 fragment (covering most of the U_S region) was located in the 28K (US2) gene. BUK-TK 900 produced small plaques on all cell lines tested in our laboratory (SK6, Vero, MDBK, 3T3). The plaque size was restored to about 70% of wild type virus plaque size when growing BUK-TK900 virus on 3T3 complementing cell line expressing PRV gE and up to 100% when cell line producing gE and gI was used. Both gE and gI genes from BUK-TK900 and from some derivative field isolates have been amplified by PCR reaction but no deletions in these genes have been found. Molecular weight of gene products differed from wild type proteins: gE was bigger than wild type gE while gI was smaller. Both proteins were correctly recognised by all tested polyclonal and monoclonal antibodies. Radioimmunoprecipitation study showed that BUK-TK900 gE and gI interact forming a complex. The whole ORF of BUK-TK900 gE was sequenced and only few point mutations were found; only two of them led to changes of amino acids in the polypeptide chain. These were: methionine at position 124 replaced by threonine and glutamine at position 162 replaced by arginine. The introduction of first of these mutations (Met to Thr) to PRV wild type strain NIA-3 resulted in 22% reduction of plaque size. This result confirms the importance of this domain of gE for its function; it was found previously by others that deletion of amino acids 125 and 126 reduced virulence and neurotropism of PRV. More changes were found in BUK-TK900 gI sequence. Over 80% of these changes were located in the terminal 1/3rd of the sequence. Some of these mutations may have significant effect on the secondary structure of gI glycoprotein. The change of the secondary structure may be responsible for the decrease of gI stability and the observed reduction of gI molecular mass.Present address: Cedi-Diagnostics B.V., Lelystad, The Netherlands.Received May 4, 2001; accepted March 18, 2003 Published online June 11, 2003     

Mutation of the herpes simplex virus 1 KOS UL45 gene reveals dose dependent effects on central nervous system growth

Summary.  The herpes simplex virus type 1 (HSV-1) UL45 gene encodes an 18 kDa virion envelope protein whose function remains unknown. Previous studies using a UL45 null mutant, UL45Δ, demonstrated that deletion of the UL45 gene altered plaque size in Vero and HeLa cells, but was not essential for replication in these cell types. The goal of this study was to determine if mutation of the UL45 gene influenced virus growth in the CNS. Two UL45 mutants, as well as a repaired revertant virus, were constructed and tested for their ability to cause encephalitis and replicate in the CNS. The UL45 mutants were not lethal when 1 × 10³ pfu were injected intracerebrally into Balb/c mice. In contrast, at inocula greater than 1 × 10³, the UL45 mutants were lethal. In vivo growth curves derived from mice inoculated intracerebrally with 1 × 10³ pfu of virus revealed that the mutants grew poorly compared to wild type or revertant viruses. These results suggest that the 18 kDa UL45 gene product is required for efficient growth in the central nervous system at low doses. We propose that the UL45 gene may play an important role under the conditions of a naturally acquired infection. Received June 23, 2001 Accepted November 1, 2001     

Homozygous deletion on the chromosomal region 5q12.3 in human lines of small-cell lung cancers

 Loss of heterozygosity at the polymorphic loci on the long arm of chromosome 5 is observed in about 80% of human small-cell lung cancer (SCLC). Absence of inactivating mutations in the APC gene on 5q14 suggests the involvement of another tumor suppressor gene. We found a homozygous deletion of sequence tagged site sequence G73332 on 5q12.3 in 2 of 12 human SCLC cell lines, Lu130 and Lu134. One copy of chromosome 5q was lost in these cell lines, and the remaining allele had a deletion in a more restricted region. A polymerase chain reaction-based analysis of yeast artificial chromosome, bacterial artificial chromosome (BAC), and lambda-phage clones narrowed the region of homozygous deletion to a fragment cloned within one BAC. Sequencing analysis revealed that a DNA fragment of approximately 25 kb was deleted interstitially, probably because of recombination through Alu repetitive sequences in Lu130 and Lu134 cells. This deletion was not detected in normal lymphocyte DNA from 98 unrelated individuals. No candidate genes, however, were detected within this region or in the adjacent 150-kb fragment. The absence of microsatellite instability and the presence of an interstitial deletion as well as gross chromosomal aberration suggest that the genomic integrity of Lu130 and Lu134 cells might possibly be affected by Alu-mediated recombination in addition to chromosomal instability. The identical breakpoints in Lu134 and Lu135 cells as well as the same genotypes at all 33 polymorphic loci examined on various chromosomes strongly suggest that these cell lines share the same genetic materials, at least in part, during the establishment or propagation of cell lines. Received: January 30, 2002 / Accepted: March 25, 2002     

Specific detection of avian pneumovirus (APV) US isolates by RT-PCR

Summary.  This report details the development of an RT-PCR assay for the specific detection of US isolates of avian pneumovirus (APV). Of the several primer pairs tested, two sets of primers derived from the matrix gene of APV were able to specifically detect the viral RNA of APV. The nucleotide sequence comparison of the PCR products of APV isolates from Minnesota suggested that these viruses were closely related to the Colorado strain of APV, but were distinct from subtypes A and B European isolates of turkey APV (turkey rhinotracheitis: TRT). This M gene-based PCR was found to be very specific and sensitive. APV as low as 8 × 10⁻⁵ TCID⁵⁰ (0.0323 μg/ml) could be detected using this assay. In addition, the two primers were able to differentiate isolates from turkeys in Minnesota. Received June 15, 1999 Accepted January 3, 2000     

Identification of the products of the equine herpesvirus type 4 gI and gE genes

Summary.  In order to identify the products of the equine herpesvirus type 4 (EHV-4) gI and gE genes, we have constructed recombinant vaccinia viruses containing the putative gI or gE genes. These recombinant viruses synthesized EHV-4 gI and gE with apparent molecular masses of 75 and 80 kDa, respectively. Antibodies raised against both recombinant viruses detected a 75 kDa gI and a 95 kDa gE in EHV-4-infected cells. The results also suggest that the EHV-4 gI and gE would form a complex like in other herpesviruses. Received October 29, 1999 Accepted January 21, 2000     

Characterisation of several heterogeneous species of defective RNAs derived from RNA 3 of cucumber mosaic virus

Summary. Preparations of double-stranded RNAs (dsRNAs) extracted from Nicotiana tabacum cv Xanthi plants infected with a subgroup IB isolate of Cucumber mosaic virus (CMV) were found to contain a heterogeneous population of defective RNAs (D-RNAs) derived from RNA 3. Characterised D-RNAs ranged in size from 1.5 to 1.9 kb and were derived either by a single in-frame deletion within the 3a or 3b genes or by means of double in-frame deletions within both genes. Also, northern blot hybridisation showed two other types of RNA derived from RNA 3: (a) RNA species of ca. 0.7 kb containing the 3′-terminus but lacking the 5′-terminus, which could be 3′-coterminal subgenomic of D-RNAs derived from the 3b gene and (b) RNA species of unknown origin of ca. 0.8 kb containing the 5′-terminus but lacking the 3′-terminus.     

Influence of homology size and polymorphism on plasmid integration in the yeast CYC1 DNA region

We studied the influence of homology size and polymorphism on the integration of circular plasmids into the yeast CYC1 region. The plasmids used also contained the URA3 gene, and the proportion of Ura⁺ transformants resulting from plasmid integration into the CYC1 region was determined by Southern-blot analysis. A size-dependent decrease in integration into the CYC1 region was observed from 858 bp to 363 bp of homology. However, with a homology size of 321, 259 or 107 bp, about 2% of the transformants still contained plasmid molecules integrated in the CYC1 region. A single point mutation in the 858-bp fragment decreased the proportion of integrations to the CYC1 gene, but the presence of additional mutations did not have a cumulative effect. For plasmids isolated in a single-stranded (ss) form, the presence of two or six point mutations did not influence integration. These results were compared with those obtained in other assays designed to study substrate requirements for homologous recombination. Received: 18 October / 15 December 1999     

A glycoprotein E gene-deleted bovine herpesvirus 1 as a candidate vaccine strain

A bovine herpesvirus 1 (BoHV-1) defective in glycoprotein E (gE) was constructed from a Brazilian genital BoHV-1 isolate, by replacing the full gE coding region with the green fluorescent protein (GFP) gene for selection. Upon co-transfection of MDBK cells with genomic viral DNA plus the GFP-bearing gE-deletion plasmid, three fluorescent recombinant clones were obtained out of approximately 5000 viral plaques. Deletion of the gE gene and the presence of the GFP marker in the genome of recombinant viruses were confirmed by PCR. Despite forming smaller plaques, the BoHV-1△gE recombinants replicated in MDBK cells with similar kinetics and to similar titers to that of the parental virus (SV56/90), demonstrating that the gE deletion had no deleterious effects on replication efficacy in vitro. Thirteen calves inoculated intramuscularly with BoHV-1△gE developed virus neutralizing antibodies at day 42 post-infection (titers from 2 to 16), demonstrating the ability of the recombinant to replicate and to induce a serological response in vivo. Furthermore, the serological response induced by recombinant BoHV-1△gE could be differentiated from that induced by wild-type BoHV-1 by the use of an anti-gE antibody ELISA kit. Taken together, these results indicated the potential application of recombinant BoHV-1 △gE in vaccine formulations to prevent the losses caused by BoHV-1 infections while allowing for differentiation of vaccinated from naturally infected animals.     

Characterization of a genomic region encoding the 32-kDa dense granule antigen of Sarcocystis muris (Apicomplexa)

Two subgenomic libraries constructed from Sarcocystis muris total DNA were screened with a cDNA probe, specific for a 32-kDa protein associated with the dense granules. Two clones reacted positively and were isolated, gDG 32/1 and gDG 32/2. Genomic clone gDG32/1 and part of clone gDG 32/2 have been sequenced. The composite nucleotide sequence of these genomic clones comprises 4.34 kb. It contains a 5′ region of 2.14 kb, a first coding region (222 bp, exon I), a noncoding region (608 bp, intron), a second coding region (660 bp, exon II), and a 3′ region of 693 bp. The upstream region shows a eukaryotic promoter structure and a consensus sequence for the 5′ and 3′ splicing sites. Thus the open reading frame (ORF DG32) coding for the 32-kDa protein of the dense granules of S. muris cyst merozoites is interrupted by an intron. To our knowledge, dg32 is the first sarcosporidian mosaic gene to be characterized. Received: 27 April 1999 / Accepted: 11 May 1999     

First known microdeletion within the Wolf-Hirschhorn syndrome critical region refines genotype-phenotype correlation

Deletions within HSA band 4p16.3 cause Wolf-Hirschhorn syndrome (WHS), which comprises mental retardation and developmental defects. A WHS critical region (WHSCR) of approximately 165 kb has been defined on the basis of 2 atypical interstitial deletions; however, genotype-phenotype correlation remains controversial, due to the large size of deletion usually involving several megabases. We report on the first known patient with a small de novo interstitial deletion restricted to the WHSCR who presented with a partial WHS phenotype consisting only of low body weight for height, speech delay, and minor facial anomalies; shortness of stature, microcephaly, seizures and mental retardation were absent. The deletion was initially demonstrated by FISH analysis, and breakpoints were narrowed with a "mini-FISH" technique using 3-5 kb amplicons. A breakpoint-spanning PCR assay defined the distal breakpoint as disrupting the WHSC1 gene within intron 5, exactly after an AluJb repeat. The proximal breakpoint was not found to be associated with a repeated sequence or a known gene. The deletion encompasses 191.5 kb and includes WHSC2, but not LETM1. Thus, manifestations attributable to this deletion are reduced weight for height, minor facial anomalies, ADHD and some learning and fine motor deficiencies, while seizures may be associated with deletions of LETM1.     

Cloning of a novel, anonymous gene from a megabase-range YAC and cosmid contig in the neurofibromatosis type 2/meningioma region on human chromosome 22q12

In order to permit detailed characterization of meningioma casesshowing deletions within chromosomal band 22q12 and furthersystematically clone genes located within this region, we establisheda genomic YAC and cosmid contig which encompasses a region inexcess of 1000 kb of 22q12. The YAC contig consists of 6 YACclones arranged Into 5 overlapping steps covering more than1100 kb. Two corresponding cosmid contigs consisting of 40 stepsof overlapping groups of cosmids encompasses 900–1000kb. This set of genomic clones provides a detailed physicalmap of this part of chromosome 22 and constitutes a basis forthe Isolation and characterization of genes that may be locatedwithin this chromosomal region. Employing the exon-amplificationmethod on two cosmids from the contig, we cloned a novel, anonymousgene, pK1.3, which potentially encodes a protein of 683 aminoacids with a predicted molecular weight of of 78.5 kD. Its 2.7kb mRNA is expressed ubiquitously. We estimated the genomicsize of this gene to 100 –150 kb, and it is located inthe Immediate centromeric vicinity of the neurofibromatosis2 (NF2) tumor suppressor gene.     

Non-homologous recombination between Alu and LINE-1 repeats caused a 430-kb deletion in the dystrophin gene: a novel source of genomic instability

Although large deletions in the dystrophin gene have been identified in more than two-thirds of Duchenne and Becker muscular dystrophy patients, the molecular mechanisms that lead to the generation of these deletions are largely unknown. Here, Alu and LINE-1 (L1) repetitive elements were shown to be present at one or other of the two ends, respectively, of a 430-kb deletion in the dystrophin gene. The breakpoint of the deletion, which stretches from exons 2 to 7, was defined more precisely by polymerase chain reaction (PCR) walking on introns 1 and 7. Finally, the region containing the breakpoint was amplified as a fragment of more than 10 kb. Sequencing of the deletion endpoint revealed the presence of an Alu sequence in intron 1, 25 kb downstream from the 3′ end of exon 1 that was joined directly to an L1 sequence in intron 7, 4.5 kb downstream from the 3′ end of exon 7. The deletion was calculated to be 430 kb. To our knowledge, this is a novel recombination event joining non-homologous Alu and L1 repeats, and is the largest known intrachromosomal deletion that is thought to involve repetitive genetic elements. Sequence characteristics around the breakpoint are discussed. Received: July 10, 2000 / Accepted: August 23, 2000     

Role of the UL28 homologue of bovine herpesvirus 1 in viral DNA cleavage and packaging

Summary.  In the aim to study the function of the bovine herpesvirus 1 (BoHV-1) UL28 protein during the replicative cycle, we characterized a UL28 deletion mutant of BoHV-1, BoHV-1 Δ UL28. Productive growth of BoHV-1 Δ UL28 was only observed in a specifically engineered complementing cell line expressing the native UL28 protein, demonstrating that UL28 is essential for virus replication. UL28 deficiency did not compromised viral protein synthesis of the late class as shown by the detection of the viral alpha gene trans-inducing factor protein encoded by UL48, a gene of the γ2 class. Southern blotting analyses of total DNA extracted from BoHV-1 Δ UL28-infected normal cells revealed that viral DNA replication was not compromised but the process of cleavage of the newly synthesized DNA was defective. Transmission electron microscopy of non-complementing BoHV-1 Δ UL28-infected cells revealed an accumulation of capsids devoid of DNA, suggesting that the DNA packaging was impaired. We conclude that the BoHV-1 UL28 protein is essential for viral replication and is necessary for the formation of mature capsid. Received October 24, 2002; accepted November 29, 2002     

Influence of ultra-long-term fatigue on the oxygen cost of two types of locomotion

The aim of this study was to examine the effects of fatigue induced by a 65-km ultramarathon on the oxygen cost of running (C_r) and cycling (C_cycl). The day before and immediately after the race, a group of nine well-trained male subjects performed two sub-maximal 4-min exercise bouts: one cycling at a power corresponding to 1.5 W · kg⁻¹ body mass on an electromagnetically braked ergometer, and one running at 11 km · h⁻¹ on a flat asphalt roadway. Before oxygen cost determinations, the subjects performed 12 “ankle” jumps at a given frequency that was fixed by an electronic metronome (2.5 Hz). From the non-fatigued to the fatigued condition, there was a significant increase in minute ventilation for both running (P < 0.01) and cycling (P < 0.0001). Significant changes were also found in respiratory exchange ratio both for running (P=0.01) and cycling (P < 0.0001). However, running and cycling differed in that C_cycl increased significantly by [mean (SD)] 24.2 (11.5)% (P < 0.001), suggesting an alteration of muscle efficiency, while C_r did not change with fatigue [186.8 (14.1) mlO₂ · kg⁻¹ · km⁻¹ vs 186.8 (18.7) mlO₂ · kg⁻¹ · km⁻¹]. In addition, contact times during hopping increased significantly from 0.173 (0.019) ms to 0.194 (0.027) ms (P < 0.01). Analysis of the factors that determine C_r indicate that the subjects modified their movement pattern in order to decrease the mechanical cost of running in such long-term fatigue conditions. Accepted: 7 August 2000     

Impact of premature stop codons on mRNA levels in infantile Sandhoff disease

Sandhoff disease is an autosomal recessive lysosomal storagedisease resulting from mutations of the HEXB gene encoding theßsubunit of ß-hexosaminidase A. Fibroblast lines fromfour patients with the infantile form of the disease were investigatedfor mutations by single strand conformation polymorphism analysisand direct sequencing of PCR products. Two of the cell lineswere homozygous for a common, 16 kb deletion of the 5’end of HEXB gene. The two other cell lines contained the 16kb deletion along with a second mutant allele generating a stopcodon: In one case a nonsense mutation, C₈₅₀—T, whichgenerated a stop codon at codon 284; and in the other, a singlebase deletion,     

Involvement of the RE V3 gene in the methylated base-excision repair system. Co-operation of two DNA polymerases, δ and Rev3p, in the repair of MMS-induced lesions in the DNA of Saccharomyces cerevisiae

The ability of four yeast DNA polymerase mutant strains to carry out the repair of DNA treated with MMS was studied. Mutation in DNA polymerase Rev3, as well as the already known mutation in the catalytic subunit of DNA polymerase δ, were both found to lead to the accumulation of single-strand breaks, which indicates defective repair. A double-mutant strain carrying mutations in DNA polymerase δ and a deletion in the REV3 gene had a complete repair defect, both at permissive (23°C) and restrictive (38°C) temperatures, which was not observed in other pairwise combinations of tested polymerase mutants. Other polymerases are not involved in the repair of exogenous DNA methylation damage, since neither mutation in the DNA polymerase ɛ, nor deletion in the DNA polymerase IV (β₇₀) gene, caused defective repair. The data obtained suggest that DNA polymerases δ and Rev3p are both necessary to perform repair synthesis in the base-excision repair of methylation damage. The results are discussed in the light of current concepts on the role of DNA polymerase Rev3 in mutagenesis. Received: 18 November / 10 December 1996