Spontaneous BHV1 recombinants in which the gI/gE/US9 region is replaced by a duplication/inversion of the US1.5/US2 region

Summary.  In a bovine herpesvirus 1 (BHV1) vaccine strain, a spontaneous BHV1 mutant (Za) was found that arose from a recombination between two isomeric forms of the BHV1 genome. In this Za mutant one end of the U_S region, containing part of the US1.5 gene, was found duplicated in an inverted orientation at the other end of the U_S region. Concurrently, a 2.7 kb deletion was found in Za that encompasses both the US8 (gE) and US9 gene. Analysis of the in vitro growth properties of a genetically modified BHV1gE⁻ mutant showed that at 11 hours post infection BHV1gE⁻ viruses were secreted ten times more efficiently than wild type virus. Using this observation we developed a protocol to enrich for spontaneous gE deletion mutants in a BHV1 field isolate and found another mutant (Rof3) with similar properties as the Za mutant. Rof3 has a duplication/inversion of the US1.5 gene and part of the US2 gene and a simultaneous 3.5 kb deletion that encompasses the US7 (gI), US8 (gE) and US9 genes. The nucleotide sequences of the recombination points of both recombinants were determined and compared. No obvious sequence similarities were found, suggesting that non-homologous recombination events led to the observed recombinations. The implications for the use of BHV1 gE deletion mutants as marker or diva vaccines are discussed. Received January 10, 1999 Accepted March 29, 1999     

Isolation and characterization of cDNA clones encoding a 32-kDa dense-granule antigen of Sarcocystis muris (Apicomplexa)

A monoclonal antibody (2F4) directed against a 32-kDa dense-granule antigen of Sarcocystis muris cyst merozoites (bradyzoites) was used to screen a lambda ZAP cDNA expression library. A clone with an insert of 1.4 kb in length (DG 32/1) was isolated. A fusion protein derived from bacteria harbouring the recombinant plasmid DG 32/1 reacted with monoclonal antibody (mAb) 2F4. Southern blot hybridization suggests that the gene is present as a single copy. On Northern blots, a single mRNA species of 1.8 kb was detected by a cDNA-derived probe. In addition, we isolated a full-length clone (DG 32/PH1) by screening the cDNA library with a non-radioactive-labelled cDNA probe. The nucleotide sequence of DG 32/PH1 comprises 1.57 kb. It contains an open reading frame of 882 bp with a coding capacity of approximately 32 kDa. The hypothetical polypeptide consists of a putative N-terminal signal peptide and the mature protein sequence. The occurrence of an N-terminal signal sequence is consistent with the observation that the 32-kDa protein of S.␣muris is secreted from the dense granules. Received: 12 December 1997 / Accepted: 15 January 1998     

A novel `two-component' protein containing histidine kinase and response regulator domains required for sporulation in Aspergillus nidulans

We have characterised a novel Aspergillus nidulans gene encoding a `two-component' signalling protein (tcsA). tcsA encodes both a histidine kinase domain and a response regulator domain similar to those found in bacterial, lower eukaryotic and plant members of the two-component family of proteins, while two PAS domains in the amino-terminal region of the predicted tcsA product may monitor the signal which regulates a tcsA histidine kinase-response regulator phosphorelay. While tcsA is nonessential for vegetative growth, cells lacking the gene are unable to produce conidia on standard Aspergillus growth media. However, tcsA is not absolutely required for production since this defect is suppressed by growth on 1 M sorbitol. Received: 23 December 1999 / 1 March 2000     

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     

<Emphasis Type="BoldItalic">Phycodnaviridae</Emphasis>– large DNA algal viruses

Summary.  Members and prospective members of the family Phycodnaviridae are large icosahedral, dsDNA (180 to 560 kb) viruses that infect eukaryotic algae. The genomes of two phycodnaviruses have been sequenced: the 331 kb genome of Paramecium bursaria chlorella virus (PBCV-1) and more recently, the 336 kb genome of the Ectocarpus siliculosus virus (EsV-1). EsV-1 has ∼ 231 protein-encoding genes whereas, the slightly smaller PBCV-1 genome has 11 tRNA genes and ∼ 375 protein-encoding genes. Surprisingly, the two viruses only have 33 genes in common, of which 17 have no counterparts in the databases. The low number of homologous genes between the two viruses can probably be attributed to their different life styles. PBCV-1 is a lytic virus that infects a unicellular, endosymbiotic freshwater green alga whereas, EsV-1 is a lysogenic virus that infects a free-living filamentous marine brown alga. Furthermore, accumulating evidence indicates that the phycodnaviruses and their genes are ancient, thus allowing significant differences to have evolved. This review briefly describes some of the biological properties of the phycodnaviruses, focusing on PBCV-1 and EsV-1, and then compares their genomes. Received December 12, 2001; accepted March 6, 2002 Published online May 25, 2002     

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.     

Genetic content of a 20.9 kb segment of human herpesvirus 6B strain Z29 spanning the homologs of human herpesvirus 6A genes U40—57 and containing the origin of DNA replication

Summary. A continuous 20.9 kb sequence from human herpesvirus 6 variant B (HHV-6B) strain Z29 (GenBank accession number L16947) is genetically colinear with a discrete segment of the human cytomegalovirus (HCMV) UL region and with HHV-6 variant A (HHV-6A). Short nucleotide sequence determinations at multiple sites within an 8.5 kb region immediately 3′ to the 20.9 kb contig revealed additional colinearity between HHV-6B, HCMV and HHV-6A. Homology studies with the predicted peptide sequences from 11 complete and 12 partial HHV-6B open reading frames (ORFs) revealed that most encode proteins conserved to varying degrees in all previously sequenced primate herpesviruses. HHV-6B homologs were identified for the HSV-1 ICP18.5, ICP8, UL52, UL24, UL25 and major capsid protein. Several HHV-6B proteins had limited amino acid similarity to their positional homologs in other herpesviruses. Each gene identified is highly homologous to its HHV-6A counterpart, including two unique HHV-6 genes predicted to encode membrane-associated glycoproteins. However, two regions of substantial divergence were noted, one spanning the origin of replication and the other encoding one of the putative HHV-6-specific glycoprotein genes. Substitutions in the latter region lead to predicted differences in reading frames and protein lengths among HHV-6 isolates. Received April 19, 1996 Accepted July 30, 1996     

The genome organization of the broad bean necrosis virus (BBNV)

Summary.  The genome of the broad bean necrosis virus Oita-isolate (BBNV-O) [RNA1 (6.0 kb), RNA2 (2.8 kb) and RNA3 (2.4 kb)] was cloned and sequenced. Computer analysis indicates that methyltransferase, helicase and RNA-dependent RNA polymerase (RdRp) motifs are present in RNA1. The viral capsid protein (CP) cistron is located at the 5′ terminal end of RNA2 and the Mr of CP (20 K) is close to that determined by SDS-PAGE analysis. An ochre codon (UAA) in the CP cistron is thought to be partially suppressed to produce a large readthrough protein. RNA3 possesses typical motifs of triple gene block proteins, which are also reported in several other plant viruses. The furovirus genome organization and phylogenetic analysis using RdRp and CP amino acid sequences suggest that BBNV is closely related to potato mop-top virus (PMTV), but is relatively distantly related to other furoviruses. The data also suggest that the genus Furovirus should be separated into several genera: the prototypical genus Furovirus, which excludes the following viruses: the PMTV group including BBNV; the beet necro- tic yellow vein virus (BNYVV) group; and the peanut clump virus (PCV) group. Received November 14, 1997 Accepted Febuary 12,1998     

Cyclin D3 gene amplification in bladder carcinoma in situ

Carcinoma in situ (CIS) is a non-papillary high-grade, potentially aggressive, and unpredictable manifestation of bladder urothelial carcinoma. The aim of this study was to assess patterns of Cyclin D3 gene amplification in Bacillus Calmette-Guerin (BCG)-treated CIS and correlate gene status with recurrence-free and progression-free survival. A sequential cohort series of 28 primary (isolated) or secondary (concomitant) bladder CIS samples in which there was enough tissue material to assess Cyclin D3 gene status by fluorescent in situ hybridization was the study group. Cyclin D3 gene amplification was present in 29% of secondary CIS; none of primary CIS samples had Cyclin D3 gene amplification. Cyclin D3 amplification was related to recurrence- (p = 0.046) and progression-free survival (p = 0.002). Type of bladder CIS (primary vs. secondary) was unrelated to recurrence- or progression-free survival in the current series. Cox’s regression analysis selected Cyclin D3 as an independent predictor of progression-free survival (p = 0.041, relative risk = 61.503, 95% confidence interval = 1.1–274.710). None of primary CIS cases recurred on follow-up; nine secondary CIS recurred and four of them progressed to invasive bladder carcinoma HG T1 (n = 1), T2b N0M0 (n = 1), T3b N1M0 (n = 1) and T4aN1M1 (n = 1). Mean recurrence ± SD (months) occurred at 19.5 ± 2.06 (95% (confidence interval (CI)), 15.5–23.6); mean progression (months) occurred at 23.8 ± 1.46 (95% (CI), 20.9–26.7). Our study suggests that Cyclin D3 gene amplification might be a predictor of aggressiveness in BCG-treated CIS.     

The family Closteroviridae revised

Summary.  Recently obtained molecular and biological information has prompted the revision of the taxonomic structure of the family Closteroviridae. In particular, mealybug-transmitted species have been separated from the genus Closterovirus and accommodated in a new genus named Ampelovirus (from ampelos, Greek for grapevine). Thus, the family now comprises three genera. Their major properties are (i) Closterovirus: type species Beet yellows virus, genome monopartite, 15.5–19.3 kb in size, a 22–25 kDa major coat protein (CP), the gene encoding the divergent CP analogue (CPd) upstream of the CP cistron, transmission by aphids, a membership of 8 definitive and 4 tentative species; (ii) Ampelo-virus: type species Grapevine leafroll virus 3, genome monopartite 16.9–19.5 kb in size, a 35–37 kDa major CP, a CPd cistron generally located downstream of the CP gene, transmission by pseudococcid and coccid mealybugs, a membership of 6 definitive and 5 tentative species; (iii) Crinivirus: type species Lettuce infectious yellows virus, genome essentially bipartite 15.3–19 kb in size, a 28–33 kDa CP, a CPd cistron downstream of the CP gene, transmission by whiteflies (Bemisia, Trialeurodes), a membership of 7 definitive and 3 tentative species. There are five unassigned species in the family.     

Anti-<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> Antibodies are Frequent in Type 1 Diabetes

Anti-Saccharomyces cerevisiae antibodies (ASCA) have been described in many autoimmune diseases in which there is an increased intestinal permeability. Also in type 1 diabetes (T1D), there is an increased intestinal permeability. Since no data are available about ASCA in T1D, we evaluated, retrospectively, the frequency of ASCA in this disease. ASCA, IgG, and IgA, were determined by ELISA in sera of 224 T1D patients in which coeliac disease has been excluded and 157 healthy control group. The frequency of ASCA (IgG or IgA) was significantly higher in T1D patients than in the control group (24.5% vs. 2.5%, p < 10⁻⁷). The same observation was found in children and in adult patients when we compare them to healthy children and blood donors group respectively. Compared to children, adult patients with T1D showed significantly higher frequencies of ASCA of any isotype (38% vs. 13.7%, p < 10⁻⁴), both ASCA IgG and IgA (12% vs. 1.6%, p = 0.002), ASCA IgG (35% vs. 9.8%, p < 10⁻⁵) and ASCA IgA (15% vs. 5.6%, p = 0.001). The frequency of ASCA was statistically higher in females of all T1D than in males (30.8% vs.17.7%, p = 0.03), in girls than in boys (22% vs.6.2%, p = 0.017), and significantly higher in men than in boys (35.7% vs. 6.2%, p < 10⁻⁴). The frequency of ASCA IgG was significantly higher than that of ASCA IgA in all T1D patients (21% vs. 9.8%, p < 0.002), in all females (26.5% vs. 10.2%, p < 0.002), in women (37.9% vs. 12%, p < 0.001). The frequency of ASCA was significantly higher in all long-term T1D than in an inaugural T1D (29% vs. 14.5%, p = 0.019). The same observation was found in adults (45.8% vs. 17.8%, p = 0.01). In long-term T1D patients, ASCA were significantly more frequent in adults than children (45.8% vs. 14.5%, p < 10⁻⁴). The frequency of ASCA IgG was significantly higher in long-term T1D than in an inaugural T1D (25.2% vs. 11.6%, p = 0.03). Patients with T1D had a high frequency of ASCA.     

Phylogenetic analyses of the matrix and non-structural genes of equine influenza viruses

Summary.  Matrix (M) and nonstructural (NS) genes of thirteen equine H3N8 and H7N7 influenza viruses were sequenced and analyzed from an evolutionary point of view. The M and NS genes of H3N8 viruses isolated between 1989 and 1993 evolved into two minor branch clusters, including isolates from Europe and the American continent, respectively. It was noteworthy to reveal that the nucleotide sequences of the M and NS genes of an earlier American strain showed highest homology to those of recent European viruses. “Frozen evolution” was observed in the M and NS genes of A/eq/LaPlata/1/88. It was also evident that the NS gene of an H7N7 virus from 1977 was very similar to that of a 1979-H3N 8 virus, while the M gene was closest phylogenetically to that of the earliest H7N7 virus isolated in 1956. Furthermore, the M2 protein of A/eq/Newmarket/1/77 virus contained a carboxyl terminal deletion of three amino acids. The evolutionary rates of the M and NS genes of H3N8 equine influenza viruses were estimated to be 5.4 × 10⁻⁴ and 5.1 × 10⁻⁴ substitutions per site per year, respectively, which were slower than those of human viruses. Received November 21, 1997 Accepted March 9, 1998     

Generation of maize cell lines containing autonomously replicating maize streak virus-based gene vectors

Summary.  Virion sense gene replacement derivatives of maize streak virus (MSV) were constructed with selectable marker expression cassettes based on the bialaphos resistance gene (bar) and the CaMV 35S promoter. The effect on replication of increasing the genomic size was tested by including: (1) the 550-bp maize adh I intron and 68-bp TMV Ω RNA leader sequences upstream of the bar genes; and (2) a fusion between the bar and E. coli glutathione reductase (gor) genes. Three recombinant viral vectors ranging in size from 2.7 kb to 4.8 kb replicated efficiently in biolistically transfected cells of suspension cultured Black Mexican sweetcorn (BMS) cells. Deletions greater than 39 bp 3′ of the stemloop sequence in the LIR adversely affected replicon release. Transformed bialaphos-resistant BMS cell lines were generated with all three vectors containing the bar gene: between 38 and 60% of cell lines contained replicating viral episomes. The replicons were structurally stable, replicated to copy numbers of over 500 per haploid genome, and were detected for more than one year after introduction. We noted significant enhancement of bar gene expression, both at the protein and RNA levels, associated with the presence of episomal vector DNA. The maize adhI intron and TMV Ω RNA leader sequences did not seem to have a significant effect on bar gene expression from replicating constructs, although expression from controls was enhanced. The results suggest that MSV-based constructs would provide a useful system for long-term gene amplification in cereal cell culture systems. Received September 4, 1998 Accepted March 4, 1999     

Complete nucleotide sequence of wheat yellow mosaic bymovirus genomic RNAs

Summary.  The complete sequences of wheat yellow mosaic bymovirus (WYMV) RNA1 and RNA2 were determined. RNA1 is 7 636 nucleotides long [excluding the 3′-poly(A)], and codes for a 269 kDa polyprotein of 2 404 amino acids which contains the capsid protein (CP) at the C terminus and seven putative non-structural proteins. RNA2 is 3 659 nucleotides long and codes for a polyprotein of 904 amino acids which contains a 28 kDa putative proteinase and a 73 kDa polypeptide. These functional proteins are arranged as in RNA1 and RNA2 of barley yellow mosaic bymovirus (BaYMV). Comparisons with the sequence reported for the 3′ half of RNA1 of wheat spindle streak mosaic bymovirus (WSSMV) from Southern France show that WYMV and WSSMV have a similar genetic organization. However, WYMV and WSSMV share only 77% amino acid sequence identity in their deduced CPs in spite of their close serological relationship, and 74% nucleotide sequence identity in their 3′ non-coding regions. Thus, the sequence data indicate that WYMV and WSSMV are not strains of the same virus, which has long been suggested, but are distinct virus species within the genus Bymovirus of the family Potyviridae. Accepted December 19, 1997 Received November 14, 1997     

A temperate phage with cohesive ends induced by mitomycin C treatment of Lactobacillus casei

Summary.  A temperate phage, named PL-2, was induced from Lactobacillus casei ATCC 27092 by mitomycin C treatment of the cells at exponential growth phase. The phage had an isometric head of 45 nm in diameter and a flexible, non-contractile tail, 150 nm long and 10 nm wide, with a sharp tip. Along the tail axis, about 40 regularly spaced striae were seen. The phage DNA had complementary cohesive ends. The restriction enzyme map of the DNA was constructed by using 13 different restriction endonucleases. The size of the DNA was 35.2 kb, 83% in size of that of phage PL-1 lytic for the same Lb. casei strain. Received December 10, 1997 Accepted March 26, 1998