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This consensus statement on the management of primary cutaneous melanoma that we present here was based on selection, discussion, review, and comparison of recent literature (including national and international guidelines). The protocols for the diagnosis, treatment, and follow-up used in the hospital centers throughout Catalonia and the Balearic Isles belonging to the Network of Catalan and Balearic Melanoma Centers were also considered. The main objective of this statement was to present the overall management of melanoma patients typically used in our region at the present time. As such, the statement was not designed to be an obligatory protocol for health professionals caring for this group of patients, and neither can it nor should it be used for this purpose. Professionals reading the statement should not therefore consider it binding on their practice, and in no case can this text be used to guarantee or seek responsibility for a given medical opinion. The group of dermatologists who have signed this statement was created 3 years ago with the aim of making our authorities aware of the importance of this complex tumor, which, in comparison with other types of cancer, we believe does not receive sufficient attention in Spain. In addition, the regular meetings of the group have produced interesting proposals for collaboration in various epidemiological, clinical, and basic applied research projects on the subject of malignant melanoma in our society.

Background and objectives:AVE5026 is a novel, hemisynthetic, ultra‐low‐molecular‐weight heparin (ULMWH), which is in clinical development for prevention of venous thromboembolism. Its unique structural features result from the highly selective depolymerization of heparin by the phosphazene base that protects the antithrombin (AT)‐binding site from destruction. In the present paper, we describe the chemical and biological characteristics of AVE5026, as well as its effects on experimental thrombosis as compared to those of the low‐molecular‐weight heparin (LMWH) enoxaparin after a single subcutaneous (s.c.) administration in certain animal models. Method and results:AVE5026 has a higher anti‐factor Xa (anti‐FXa) activity (∼160 U mg−1) along with a catalytic anti‐thrombin (anti‐FIIa) activity (∼2 U mg−1) as a result of its structure being strongly enriched in specific AT‐binding oligosaccharides. In human plasma, potent inhibition of thrombin generation by AVE5026 was closely related to its anti‐FXa activity. In a rat venous thrombosis model, AVE5026 showed a dose‐dependent antithrombotic activity comparable to that of enoxaparin (ED50‐AVE5026 = 1.6 mg kg−1, ED50‐enoxaparin = 2.8 mg kg−1). Interestingly, non‐occlusive venous thrombosis in rabbits was inhibited by an ED50 of 0.1 mg kg−1AVE5026, whereas 0.316 mg kg−1enoxaparin was not active. In a canine model, similarly to enoxaparin (ED50 = 1.3 mg kg−1), AVE5026 dose‐dependently inhibited arterial thrombosis (ED50 = 2.0 mg kg−1). At equipotent doses, AVE5026 did not affect bleeding parameters, whereas enoxaparin showed increased hemorrhage in rats, rabbits and dogs. Conclusion:These unique structural attributes distinguish AVE5026 from the LMWH class. Based on these data in well‐established arterial and venous thrombosis models, AVE5026 could represent a valuable alternative in thrombosis prevention with an improved benefit‐risk profile as compared to that of enoxaparin.

Baculovirus genomes are covalently closed circles of double stranded-DNA varying in size between 80 and 180 kilobase-pair. The genomes of more than fourty-one baculoviruses have been sequenced to date. The majority of these (37) are pathogenic to lepidopteran hosts; three infect sawflies (Hymenoptera) and one has a mosquito host (Diptera). With this information, general patterns of genome structure and gene content became apparent. Baculovirus open reading frames are tightly packed with minimal intergenic regions and the coding sequences are almost equally distributed over both strands. Baculovirus genes form single transcription units, with early and late transcribed ORFs scattered along the genome. A set of twenty nine core genes is conserved and therefore is characteristic for baculoviruses. Most baculovirus genomes contain multiple homologous regions with repeated sequences and often palindromic motifs, which play a crucial role as enhancers of early transcription and most likely in viral DNA replication. Baculovirus genomes have a certain degree of plasticity, as evidenced from the genomic variations within virus isolates from the field. Recombination events and transposon insertions appear to play a role in the uptake of new genes from co-infecting viruses or from the insect host. This review deals with the structural and functional properties of baculovirus genomes including both conserved and variable genes.

AbstractIn this study the voice characteristics of normal male and female speakers are compared to those of two groups of patients with unilateral vocal fold paralysis. In order to enhance phonation, the patients in the first group compensate for the adduction deficiency which results from paralysis. The patients in the second group do not use compensatory strategies. Sustained vowels [i:, a:, u:] were produced by the speakers and scored for roughness, breathiness and hoarseness (RBH) by 8 raters. Although interrater agreement for RBH scores is only moderate on average, these percepts make consistent distinctions between the three speaker groups. Consistent but different distinctions are made between the three speaker groups for male and female speakers. The results show that male and female speakers should not be pooled in experimental studies of the pathological voice. Our results also indicate that female patients with a compensated unilateral vocal fold paralysis cannot be clinically evaluated solely on the basis of perception, because their voices cannot be distinguished from normal, healthy female speakers, despite their physiological impairment. The group distinctions made on the basis of RBH scores are supported by differences in the acoustic parameters which are derived by automatic analysis of the sustained vowels. Despite identical group distinctions for RBH scores and acoustic parameters, the acoustic basis of the percepts is not straightforward. Different acoustic predictors of the percepts were found for male compared to female speakers. Additionally, interrater differences point towards the presence of perceptual trading relations.Copyright © 2004 S. Karger AG, Basel

Theileria parva is an intracellular protozoan parasite that causes East Coast fever, a severe lymphoproliferative disease in cattle. Previous attempts to produce recombinant sporozoite surface antigen (p67) in bacterial or insect cells for vaccine purposes have not resulted in a correctly folded protein. Here, we report the expression of N- and C-terminal domains of p67 fused to the baculovirus envelope glycoprotein GP64 by cloning the appropriate p67 cDNA segments between the signal sequence and the major portion of GP64. To further advance the generation of such recombinants, existing surface display techniques were combined with bacmid technology. Chimeric proteins were present on the surface of budded viruses as judged by immunogold labelling and were exposed on the surface of insect cells, as concluded from immunofluorescence studies of infected, non-fixed insect cells. In non-denaturing dot blot experiments, a strong reaction was obtained between monoclonal TpM12 and baculovirus particles displaying the p67N-GP64 chimeric protein. This antibody, raised against native p67, also specifically recognized the surface of recombinant-infected cells. Apparently, a more native conformation was achieved than when p67 was expressed in E.coli or in conventional baculovirus expression systems. The baculovirus surface expression system, therefore, provides an improved way of expressing this T.parva sporozoite surface protein.

The generation of deletion mutants, including defective interfering viruses, upon serial passage of Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) in insect cell culture has been studied. Sequences containing the non-homologous region origin of DNA replication (non-hr ori) became hypermolar in intracellular viral DNA within 10 passages in Se301 insect cells, concurrent with a dramatic drop in budded virus and polyhedron production. These predominant non-hr ori-containing sequences accumulated in larger concatenated forms and were generated de novo as demonstrated by their appearance and accumulation upon infection with a genetically homogeneous bacterial clone of SeMNPV (bacmid). Sequences were identified at the junctions of the non-hr ori units within the concatemers, which may be potentially involved in recombination events. Deletion of the SeMNPV non-hr ori using RecE/RecT-mediated homologous ET recombination in Escherichia coli resulted in a recombinant bacmid with strongly enhanced stability of virus and polyhedron production upon serial passage in insect cells. This suggests that the accumulation of non-hr oris upon passage is due to the replication advantage of these sequences. The non-hr ori deletion mutant SeMNPV bacmid can be exploited as a stable eukaryotic heterologous protein expression vector in insect cells.

ABSTRACTGP64, the major envelope glycoprotein of budded virions of the baculovirus Autographa californicamulticapsid nucleopolyhedrovirus (AcMNPV), is involved in viral attachment, mediates membrane fusion during virus entry, and is required for efficient virion budding. Thus, GP64 is essential for viral propagation in cell culture and in animals. Recent genome sequences from a number of baculoviruses show that only a subset of closely related baculoviruses have gp64genes, while other baculoviruses have a recently discovered unrelated envelope protein named F. F proteins from Lymantria dispar MNPV (LdMNPV) and Spodoptera exigua MNPV (SeMNPV) mediate membrane fusion and are therefore thought to serve roles similar to that of GP64. To determine whether F proteins are functionally analogous to GP64 proteins, we deleted the gp64gene from an AcMNPV bacmid and inserted F protein genes from three different baculoviruses. In addition, we also inserted envelope protein genes from vesicular stomatitis virus (VSV) and Thogoto virus. Transfection of the gp64-null bacmid DNA into Sf9 cells does not generate infectious particles, but this defect was rescued by introducing either the F protein gene from LdMNPV or SeMNPV or the G protein gene from VSV. These results demonstrate that baculovirus F proteins are functionally analogous to GP64. Because baculovirus F proteins appear to be more widespread within the family and are much more divergent than GP64 proteins, gp64may represent the acquisition of an envelope protein gene by an ancestral baculovirus. The AcMNPV pseudotyping system provides an efficient and powerful method for examining the functions and compatibilities of analogous or orthologous viral envelope proteins, and it could have important biotechnological applications.

ABSTRACTThe generation of deletion mutants, including defective interfering viruses, upon serial passage of Spodoptera exiguamulticapsid nucleopolyhedrovirus (SeMNPV) in insect cell culture has been studied. Sequences containing the non-homologous region origin of DNA replication (non-hr ori) became hypermolar in intracellular viral DNA within 10 passages in Se301 insect cells, concurrent with a dramatic drop in budded virus and polyhedron production. These predominant non-hr ori-containing sequences accumulated in larger concatenated forms and were generated de novo as demonstrated by their appearance and accumulation upon infection with a genetically homogenous bacterial clone of SeMNPV (bacmid). Sequences were identified at the junctions of the non-hr oriunits within the concatemers, which may be potentially involved in recombination events. Deletion of the SeMNPV non-hr oriusing RecE/RecT-mediated homologous ET recombination in Escherichia coliresulted in a recombinant bacmid with strongly enhanced stability of virus and polyhedron production upon serial passage in insect cells. This suggests that the accumulation of non-hr oris upon passage is due to the replication advantage of these sequences. The non-hr orideletion mutant SeMNPV bacmid can be exploited as a stable eukaryotic heterologous protein expression vector in insect cells.

Precise measurements of pyroelectric coefficient, the thermal deformation and linear birefringence changes in the Li 2 Ge 7 O 15 were performed in a wide temperature range. Temperature behaviour of spontaneous deformation and linear birefringence increment did not reflect the one demonstrated by the polarisation. Critical behaviour of the linear birefringence increment at the phase transition region will be discussed and compared with earlier reports.

Newly discovered phase transition at 233.5 K in the Li 2 TiGeO 5 crystal was studied with various physical methods. Results of dielectric, dilatometric, calorimetric, linear birefringence measurements, ferroelastic domain structure observations, the Brillouin scattering studies demonstrated a ferroelastic character of the second order transition, of 4/mmmFmmm Aizu type.

ABSTRACTThe Spodoptera exiguamulticapsid nucleopolyhedrovirus (SeMNPV) Se8gene was recently shown to encode the viral envelope fusion (F) protein. A 60-kDa C-terminal subunit (F1) of the 76-kDa primary translation product of this gene was found to be the major envelope protein of SeMNPV budded virus (BV) (W. F. J. IJkel, M. Westenberg, R. W. Goldbach, G. W. Blissard, J. M. Vlak, and D. Zuidema, Virology 275:30–41, 2000). A specific inhibitor was used to show that furin is involved in cleavage of the precursor envelope fusion (F0) protein. BV produced in the presence of the inhibitor possesses the uncleaved F0protein, while an F protein with a mutation in the furin cleavage site was translocated to the plasma membrane but lost its fusogenic activity. These results indicate that cleavage of F0is required to activate the SeMNPV F protein and is necessary for BV infectivity. Specific antibodies against F1and against the putative N terminus (F2) of the primary translation product were used to show that the F protein is BV specific and that BVs contain both the 60- (F1) and 21-kDa (F2) cleavage products. In nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis both subunits migrate as a single 80-kDa protein, indicating that the subunits remain associated by a disulfide linkage. In addition, the presence of the F protein predominately as a monomer suggests that disulfide links are not involved in oligomerization. Thus, the envelope fusion protein from group II nucleopolyhedroviruses of baculoviruses has properties similar to those of proteins from a number of vertebrate viruses.

ABSTRACTSeveral phylogenetic methods based on whole genome sequence data were evaluated using data from nine complete baculovirus genomes. The utility of three independent character sets was assessed. The first data set comprised the sequences of the 63 genes common to these viruses. The second set of characters was based on gene order, and phylogenies were inferred using both breakpoint distance analysis and a novel method developed here, termed neighbor pair analysis. The third set recorded gene content by scoring gene presence or absence in each genome. All three data sets yielded phylogenies supporting the separation of the Nucleopolyhedrovirus(NPV) andGranulovirus(GV) genera, the division of the NPVs into groups I and II, and species relationships within group I NPVs. Generation of phylogenies based on the combined sequences of all 63 shared genes proved to be the most effective approach to resolving the relationships among the group II NPVs and the GVs. The history of gene acquisitions and losses that have accompanied baculovirus diversification was visualized by mapping the gene content data onto the phylogenetic tree. This analysis highlighted the fluid nature of baculovirus genomes, with evidence of frequent genome rearrangements and multiple gene content changes during their evolution. Of more than 416 genes identified in the genomes analyzed, only 63 are present in all nine genomes, and 200 genes are found only in a single genome. Despite this fluidity, the whole genome-based methods we describe are sufficiently powerful to recover the underlying phylogeny of the viruses.

White spot syndrome virus (WSSV) is at present a major scourge to worldwide shrimp cultivation. We have determined the entire sequence of the double-stranded, circular DNA genome of WSSV, which contains 292,967 nucleotides encompassing 184 major open reading frames (ORFs). Only 6% of the WSSV ORFs have putative homologues in databases, mainly representing genes encoding enzymes for nucleotide metabolism, DNA replication, and protein modification. The remaining ORFs are mostly unassigned, except for five, which encode structural virion proteins. Unique features of WSSV are the presence of a very long ORF of 18,234 nucleotides, with unknown function, a collagen-like ORF, and nine regions, dispersed along the genome, each containing a variable number of 250-bp tandem repeats. The collective information on WSSV and the phylogenetic analysis on the viral DNA polymerase suggest that WSSV differs profoundly from all presently known viruses and that it is a representative of a new virus family.

White spot syndrome virus (WSSV) is a large DNA virus infecting shrimp and other crustaceans. The virus particles contain at least five major virion proteins, of which three (VP26, VP24, and VP15) are present in the rod-shaped nucleocapsid and two (VP28 and VP19) reside in the envelope. The mode of entry and systemic infection of WSSV in the black tiger shrimp, Penaeus monodon,and the role of these proteins in these processes are not known. A specific polyclonal antibody was generated against the major envelope protein VP28 using a baculovirus expression vector system. The VP28 antiserum was able to neutralize WSSV infection of P. monodonin a concentration-dependent manner upon intramuscular injection. This result suggests that VP28 is located on the surface of the virus particle and is likely to play a key role in the initial steps of the systemic WSSV infection in shrimp.

Understanding the molecular basis of the distinct biological properties of Spodoptera exiguamulticapsid nucleopolyhedrovirus (SeMNPV), such as its narrow host range and high virulence, requires detailed information on the temporal expression and subcellular localization of SeMNPV gene products. The expression of two unique SeMNPV ORFs, 116 (Se116) and 117 (Se117), which show 45% amino acid similarity, was analyzed. Se116 and Se117 were expressed both in cultured cells and in larvae of S. exiguaas polyadenylated transcripts of 0.80 and 0.75 kb, respectively. These transcripts initiated from ATCA(G/T)T promoter motifs, commonly found for baculovirus early genes. Se116 transcripts were detected with increasing abundance from 8 to 48 h p.i., whereas Se117 transcripts were present from 4 h p.i. and most abundantly at 24 h p.i. Western blot analysis of infected Se301 cells revealed 27- and 23-kDa proteins for Se116 and Se117, respectively. C-terminal GFP–fusion proteins of Se116 and Se117 were primarily localized in the nucleus of Se301 cells. When Se301 cells were infected with SeMNPV, both GFP–fusion proteins were localized in the virogenic stroma of the nucleus. While the function of the Se116 protein is still enigmatic, the Se117 protein appeared to be a structural protein associated with nucleocapsids of occlusion-derived SeMNPV virions but not of budded virus.

Defective interfering baculoviruses (DIs) lack considerable portions of the genome, interfere with the replication of helper virus, and cause the so-called “passage-effect” during serial passaging in insect cells and in bioreactor configurations. We investigated their origin by (nested) PCR and demonstrated that DIs lacking approximately 43% (d43) of their DNA are present in low-passage Autographa californicamulticapsid nucleopolyhedrovirus (AcMNPV)-E2 virus stocks and in polyhedra, but not in the authentic AcMNPV isolate obtained prior to passage in cell culture. To investigate whether DIs are rapidly generated de novoin Sf21 insect cells, a genetically homogeneous AcMNPV bacmid was serially passaged, resulting in the generation of d43 DIs within two passages. AT-rich sequences of up to 66 nucleotides of partly unknown origin were found at the deletion junctions in the d43 DI genomes. These data suggest that the rapid generation of DIs is an intrinsic property of baculovirus infection in insect cell culture and involves several recombination steps.

Antagonists of the platelet fibrinogen receptor (GP IIb/IIIa receptor) are expected to be a promising new class of antithrombotic agents. The binding of fibrinogen to the fibrinogen receptor depends on an Arg-Gly-Asp-Ser (RGDS) tetrapeptide recognition motif. Structural modifications of the RGDS lead have led to the discovery of a non-peptide RGD mimetic GP IIb/IIIa antagonist 44 (S 1197). Compound 44 inhibited, in a dose dependent and reversible manner, human and dog platelet aggregation as well as ¹²⁵I-fibrinogen binding to ADP-activated human gel filtered platelets and isolated GP IIb/IIIa with Ki values of 9 nM and 0.17 nM, respectively. A pharmacophore mapping procedure with QXP and a 3D-QSAR analysis applying the GRID/GOLPE methodology yielded a stable, rather predictive model and revealed structural features which are important for binding. Hydrophobic substitutions both at the hydantoin nucleus and at the C-terminus increase the affinity toward the fibrinogen receptor. The crystalline ethyl ester prodrug 48 (HMR 1794) is an orally active antithrombotic agent which is a promising drug candidate for the treatment of thrombotic diseases in humans.

White spot syndrome virus (WSSV) is a virus infecting shrimp and other crustaceans, which is unclassified taxonomically. A 2193 bp long open reading frame, encoding a putative protein kinase (PK), was found on a 8.4 kb EcoRI fragment of WSSV proximal to the gene for the major envelope protein (VP28). The identified PK shows a high degree of homology to other viral and eukaryotic PK genes. Homology in the catalytic domains suggests that this PK is a serine/threonine protein kinase. All of the conserved PK domains are present in the WSSV PK gene product and this allowed the alignment with PK proteins from other large DNA viruses, which encode one or more PK proteins. An unrooted parsonimous phylogenetic tree was constructed and indicated that the PK gene is well conserved in all DNA virus families and hence can be used as a phylogenetic marker. Baculoviruses to date contain only a single PK gene, which is present in a separate well bootstrap-supported branch in the tree. The WSSV PK is not present in the baculovirus clade and therefore is clearly separated phylogenetically from the baculovirus PK genes. Furthermore, the WSSV PK gene does not share a most recent common ancestor with any known PK gene from other viruses. This provides further and independent evidence for the unique position of WSSV in a newly proposed genus named Whispovirus.

The causative agent of white spot syndrome (WSS) is a large double-stranded DNA virus, WSSV, which is probably a representative of a new genus, provisionally called Whispovirus. From previously constructed WSSV genomic libraries of a Taiwan WSSV isolate, clones with open reading frames (ORFs) that encode proteins with significant homology to the class I ribonucleotide reductase large (RR1) and small (RR2) subunits were identified. WSSV rr1and rr2potentially encode 848 and 413 amino acids, respectively. RNA was isolated from WSSV-infected shrimp at different times after infection and Northern blot analysis with rr1- and rr2-specific riboprobes found major transcripts of 2.8 and 1.4 kb, respectively. 5′ RACE showed that the major rr1transcript started at a position of −84 (C) relative to the ATG translational start, while transcription of the rr2gene started at nucleotide residue −68 (T). A consensus motif containing the transcriptional start sites for rr1and rr2was observed (TCAc/tTC). Northern blotting and RT-PCR showed that the transcription of rr1and rr2started 4–6 h after infection and continued for at least 60 h. The rr1and rr2genes thus appear to be WSSV “early genes.”

Dose– and time–mortality relationships of baculoviruses in pest insects are important for the determination of effective spraying regimes. A series of experiments with Autographa californicamulticapsid nucleopolyhedrovirus (AcMNPV) and Spodoptera exiguaMNPV (SeMNPV) against synchronized populations of S. exigualarvae in greenhouse chrysanthemum was conducted. Dose– and time–mortality relationships of different virus concentrations and S. exiguatarget stages were determined and the area foliage consumption was measured. Crop injury was greatly reduced when S. exiguawere controlled as second or third instar larvae, whereas virus applications against fourth instar larvae could not prevent considerable crop injury, even at high concentrations. SeMNPV was approximately 10 times as infectious as AcMNPV when applied on greenhouse chrysanthemum. The relative virulence of AcMNPV and SeMNPV corresponded reasonably well with previously published laboratory bioassay data. SeMNPV killed second and fourth instar S. exigualarvae approximately 12 h faster than did AcMNPV in chrysanthemum, but no difference in speed of action was found for third instar larvae. The relative speed of action of AcMNPV and SeMNPV determined in chrysanthemum and in laboratory bioassays did not correspond for third instar S. exigualarvae; laboratory bioassay data can therefore not simply be extrapolated to the crop level.

The insecticidal efficacy of baculoviruses can be enhanced by engineering the viral genome to express proteins that disrupt the physiology of the host insect. Here we describe the development of a genetically engineered Autographa californicamulticapsid nucleopolyhedrovirus (AcMNPV) which expresses a modified form of juvenile hormone esterase (JHE). Previously, two viruses expressing different modified JHEs were found to have a greater insecticidal effect on larvae of Trichoplusia niand Heliothis virescensthan a virus expressing wild-type JHE. To study a possible synergistic effect, the distinct mutations in the modified JHEs were combined in a new JHE construct. Two lysine residues were replaced with arginine residues to reduce the efficiency of lysosomal targeting (JHE-KK) and the catalytic serine was replaced with glycine, which eliminated catalytic activity (JHE-SG). The modified JHE, JHE-KSK, was expressed in a recombinant baculovirus, AcJHE-KSK. Larvae of H. virescensinfected with this recombinant virus caused 44% less feeding damage to lettuce than larvae infected with the wild-type AcMNPV. However, AcJHE-KSK did not have significantly improved insecticidal properties over the parent viruses AcJHE-KK and AcJHE-SG, suggesting that the separate mutations have no major synergistic effect. Infection with a control recombinant baculovirus expressing JHE with the same lysine to arginine conversions and in which a catalytic histidine was converted to lysine (AcJHE-KHK) did not reduce feeding damage compared with that caused by larvae infected with AcMNPV.

The entry mechanism of Spodoptera exiguamulticapsid nucleopolyhedrovirus (SeMNPV), a group II NPV, in cultured cells was examined. SeMNPV budded virus (BV) enters by endocytosis as do the BVs of the group I NPVs, Autographa californica(Ac) MNPV and Orgyia pseudotsugata(Op) MNPV. In group I NPVs, upon infection acidification of the endosome triggers fusion of the viral and endosomal membrane, which is mediated by the BV envelope glycoprotein GP64. However, the SeMNPV genome lacks a homolog of GP64 envelope fusion protein (EFP). A functional homolog of the OpMNPV GP64 EFP was identified in SeMNPV ORF8 (Se8; 76 kDa) and appeared to be the major BV envelope protein. Surprisingly, a 60-kDa cleavage product of this protein is present in the BV envelope. A furin-like proprotein convertase cleavage site (R-X-K/R-R) was identified immediately upstream of the N-terminus of the mature Se8 protein and this site was also conserved in the Lymantria dispar(Ld) MNPV homolog (Ld130) of Se8. Syncytium formation assays showed that Se8 and Ld130 alone were sufficient to mediate membrane fusion upon acidification of the medium. Furthermore, C-terminal GFP-fusion proteins of Se8 and Ld130 were primarily localized in the plasma membrane of insect cells. This is consistent with their fusogenic activity and supports the conclusion that the Se8 gene product is a functional homolog of the GP64 EFP.

The Helicoverpa armigerasingle-nucleocapsid nucleopolyhedrovirus (HearNPV) has been registered and is commercially produced in China as a biopesticide to control the bollworm in cotton. However, the virus has a relatively slow speed of action. To improve its efficacy, recombinant HearNPVs were generated by deleting the ecdysteroid UDP-glucosyltransferase (egt) gene (HaCXW1 and HaLM2) or by inserting the insect-specific toxin gene AaIT in the egtlocus (HaCXW2) of HearNPV using conventional recombination strategies in insect cell culture. The various recombinants remained genetically stable when cultured in HzAM1 insect cells. Bioassay data showed a significant reduction in the time required for all HearNPV recombinants to kill second instar H. armigeralarvae. The LT50of the egtdeletion recombinants HaCXW1 and HaLM2 was about 27% faster than that of wild-type HearNPV. The largest reduction in LT50was achieved by inserting the gene for the insect-specific neurotoxin, AaIT, in the egtlocus, giving a reduction in LT50of 32% compared to wild-type HearNPV. The ability to genetically improve the properties of HearNPV as a biopesticide provides a further opportunity to develop this virus into a commercially viable product to control the bollworm in China.

White Spot Syndrome Virus (WSSV) is an invertebrate virus, causing considerable mortality in shrimp. Two structural proteins of WSSV were identified. WSSV virions are enveloped nucleocapsids with a bacilliform morphology with an approximate size of 275 × 120 nm, and a tail-like extension at one end. The double-stranded viral DNA has an approximate size 290 kb. WSSV virions, isolated from infected shrimps, contained four major proteins: 28 kDa (VP28), 26 kDa (VP26), 24 kDa (VP24), and 19 kDa (VP19) in size, respectively. VP26 and VP24 were found associated with nucleocapsids; the others were associated with the envelope. N-terminal amino acid sequences of nucleocapsid protein VP26 and the envelope protein VP28 were obtained by protein sequencing and used to identify the respective genes (vp26and vp28) in the WSSV genome. To confirm that the open reading frames of WSSV vp26(612) and vp28(612) are coding for the putative major virion proteins, they were expressed in insect cells using baculovirus vectors and analyzed by Western analysis. A polyclonal antiserum against total WSSV virions confirmed the virion origin of VP26 and VP28. Both proteins contained a putative transmembrane domain at their N terminus and many putative N- and O-glycosylation sites. These major viral proteins showed no homology to baculovirus structural proteins, suggesting, together with the lack of DNA sequence homology to other viruses, that WSSV may be a representative of a new virus family, Whispoviridae.

Virulence and speed of action, as related to dose, are important effectiveness-determining properties of insect-pathogenic biocontrol agents. We used the droplet-feeding bioassay to compare dose responses between two wild-type baculoviruses, Autographa californicamulticapsid nucleopolyhedrovirus (AcMNPV) and Spodoptera exiguaMNPV (SeMNPV), and three deletion mutants of AcMNPV in S. exigualarvae. In each mutant one gene was deleted by genetic engineering: pp34,coding for the polyhedral membrane; egt,coding for ecdysteroid UDP–glucosyltransferase; or p10,coding for fibrillar structures in infected insect cells. SeMNPV had the lowest median lethal dose (LD50) as well as the highest speed of action (LT50) of all viruses investigated. In our comparative bioassays the only significant effect of gene deletions in AcMNPV was a slightly lower speed of action for the p10deletion mutant. Otherwise, wild-type and recombinant AcMNPVs had similar biological activities. Our results suggest, in contrast to what is generally assumed, that gene deletions in AcMNPV for improved insecticidal activity should be critically assessed in each host system prior to further implementation as a control agent. Insertion of foreign genes coding for entomotoxins is less questionable and more promising in this respect.

The Manx gene is required for the development of the tail and other chordate features in the ascidian tadpole larva. To determine the structure of the Manx gene, we isolated and sequenced genomic clones from the tailed ascidian Molgula oculata. The Manx gene contains 9 exons and encodes both major and minor Manx mRNAs, which differ in the length of their 5' untranslated regions. The coding region of the single-copy bobcat gene, which encodes a DEAD-box RNA helicase, is embedded within the first Manx intron. The organization of the bobcat and Manx transcription units was determined by comparing genomic and cDNA clones. The Manx-bobcat gene locus has an unusual organization in which a non-coding first exon is alternatively spliced at the 5' end of two different mRNAs. The bobcat and Manx genes are expressed coordinately during oogenesis and embryogenesis, but not during spermatogenesis, in which bobcat mRNA accumulates independently of Manx mRNA. Similar to Manx, zygotic bobcat transcripts accumulate in the embryonic primordia responsible for generating chordate features, including the dorsal neural tube and notochord, are downregulated during embryogenesis in the tailless species Molgula occulta and are upregulated in M. occulta X M. oculata hybrids, which restore these chordate features. Antisense experiments indicate that zygotic bobcat expression is required for development of the same suite of chordate features as Manx. The results show that the Manx-bobcat gene complex has a role in the development of chordate features in ascidian tadpole larvae.

Antibodies of various immunoglobulin classes to different cow's milk proteins were studied with the fluorescent immunosorbent test in 601 newborns, infants, children and adults (A). The antibody levels, expressed as the geometric mean (gm) of four antibody titres to casein, ß-lactoglobulin, a-lactalbumin and bovine serum albumin, showed a clear dependence on age. They were compared with the antibody levels in children with cow's milk protein intolerance (C), other gastrointestinal disorders (B) and coeliac disease (D). The 20 children with cow's milk protein intolerance clearly differed (significance level 2×10-11) from those of the two control groups (A, B) insofar as the criterion adopted was not the titre against a single protein but the gm of the four antibody titres, and insofar as allowance was made for the age of the patients.

The protozoan parasiteBabesia equi, a causative agent of equine piroplasmosis, was continuously cultivated in horse erythrocytes. The parasites were isolated from a carrier horse at a time when no parasite was detected in a thin blood smear. The culture medium consisted of modified medium 199 supplemented with 40% non-heat-inactivated horse serum in a humidified atmosphere containing 5% CO2, 2% O2, and 93% N2 at 37° C. Parasites were detected after 2 days in culture. When the percentage of parasitized erytrrocytes (PPE) reached 1%, the cultures were transferred into a humidified atmosphere of 5% CO2 in air. After 7 days the cultures were split at a ratio of 1:2, and after another 5 days they were split at a ratio of 1:4. From them on, cultures were split at a ratio of 1:4 routinely at 2-day intervals. The PPE ranged between 10% and 25%. Supplementation with hypoxanthine was essential for the initiation and propagation of cultures. In established cultures, hypoxanthine could be replaced by equimolar concentrations of adenosine or guanosine. Parasites from cultures could be cryopreserved and resuscitated. Cultures were maintained for more than 300 days.

A full-length human creatine kinase B (B-CK) cDNA was used to produce a recombinant baculovirus (AcDZ1-BCK). Sf9 cells infected with this recombinant expressed a homodimeric protein composed of 43 kDa subunits which, under optimal conditions, formed up to 30% of the total soluble cellular protein. Upon analysis by PAGE, zymogram assay and gel filtration chromatography the recombinant protein behaved like authentic dimeric human BB-CK protein. Studies with a newly produced monoclonal antibody (CK-BYK/21E10) directed against an epitope in the N-terminus of the protein confirmed the identity of the product. The recombinant BB-CK protein was purified to over 99% homogeneity from the total protein extract of AcDZ1-CKB infected cells in one single step involving anion exchange column chromatography on MonoQ in FPLC. Dialysed protein had a specific activity of 239 U/mg protein.

The antiinflammatory activity of twelve medicinal plants used against skin disorders were tested in different experimental models of topical inflammation and one in vitro inhibitory test against phospholipase A2 (PLA2) from Naja naja venom. Forsythia suspensa was the most active species on the arachidonic acid (AA) topical test. This last species together with Astragalus membranaceus and Ranunculus sceleratus were the most active on the 12-O-tetradecanoylphorbol-13-acetate (TPA) acute ear oedema test. Scrophularia auriculata was the most active on multiple topical applications of TPA and on the oxazolone-induced delayed type hypersensitivity (DTH). Santolina chamaecyparissus was the only species that inhibited PLA2 in vitro. © 1998 John Wiley & Sons, Ltd.

Summary To prevent nosocomial rotavirus infections in hospitalized children with various non-gastrointestinal diseases, 30 children (mean age five months) received 200 ml of fresh human milk per day in addition to the normal diet for their age. A matched group of children on formula diet served as a control. Fecal samples were routinely screened for rotavirus by a commercial ELISA test. In stools containing rotavirus, the virus RNA segments were analysed by gel electrophoresis to identify the different rotavirus strains. Clinical symptoms were recorded daily and quantified by a score system. Human milk had no effect on the frequency of nosocomial rotavirus infections: ten infected children were fed with human milk and seven were not. However, the severity of the clinical symptoms was clearly reduced: the mean score of clinical symptoms was only half as great and the number of mild or asymptomatic infections was doubled in the group receiving fresh human milk.

Trials with live cytomegalovirus (TOWNE 125 strain), which was attenuated by 125 passages exclusively on WI-38 cells, were done in adult volunteers. No virus take occured after oral/nasal application. When 103TCD50was given intramuscularly an IgG antibody response was detected at four weeks in all of ten volunteers tested by immunofluorescence; an IgM response was found in seven. Only mild local side-effects and relative lymphocytosis were observed. No virus excretion was found. Many questions remain to be answered by further trials before a cytomegalovirus vaccine can be given to adolescent girls. Bei erwachsenen Freiwilligen wurden Immunisierungsversuche mit lebenden Zytomegalieviren, welche durch 125 Passagen ausschließlich auf WI-38 Zellen attenuiert worden waren (TOWNE 125 Stamm), durchgeführt. Orale/nasale Applikation induzierte keine Antikörperbildung. Fluoreszierende IgG-Antikörper konnten bei allen zehn sero-negativen Probanden, welche die attenuierten Zytomegalieviren in einer Dosis von 103TCD50intramuskulär erhielten, vier Wochen nach Immunisierung gefunden werden. Zum Auftreten von IgM-Antikörpern kam es bei sieben der zehn Sero-Negativen. Klinisch traten nur geringfügige Lokalreaktionen und eine relative Lymphozytose auf. Es konnte keine Virusausscheidung nachgewiesen werden. Verschiedene bis jetzt noch ungeklärte Fragen müssen zuerst durch weitere „Impfversuche“ an Freiwilligen geklärt werden, bevor an eine generelle Durchimpfung weiblicher Adoleszenten gegen Zytomegalie gedacht werden kann.

The amino acid sequences necessary for entomocidal activity of the CryIA(b) protoxin of Bacillus thuringiensis were determined. Introduction of stop codons behind codons Arg601, Phe604 or Ala607 showed that amino acid residues C-terminal to Ala607 are not required for insecticidal activity and that activation by midgut proteases takes place distal to Ala607. The two shortest polypeptides, deleted for part of the highly conserved ß-strand, were prone to proteolytic degradation, explaining their lack of toxicity. Apparently, this ß-strand is essential for folding of the molecule into a stable conformation. Proteolytic activation at the N-terminus was investigated by removing the first 28 codons, resulting in a translation product extending from amino acid 29 to 607. This protein appeared to be toxic not only to susceptible insect larvae such as Manduca sexta and Heliothis virescens, but also to Escherichia coli cells. An additional mutant, encoding only amino acid residues 29–429, encompassing the complete putative pore forming domain, but lacking a large part of the receptor-binding domain, was similarly toxic to E. coli cells. This suggests a role for the N-terminal 28 amino acids in rendering the toxin inactive in Bacillus thuringiensis, and indicates that the cytolytic potential of the pore forming domain is only realized after proteolytic removal of these residues by proteases in the insect gut. In line with this hypothesis are results obtained with a mutant protein in which Arg28 at the cleavage site was replaced by Asp. This substitution prevented the protein from being cleaved by trypsin in vitro, and reduced its toxicity to M. sexta larvae.

Summary Pre- and postvaccination sera from 32 children aged 1.5 to 2 years vaccinated with two different mumps vaccines were available for the determination of mumps antibodies. Five different tests for mumps antibodies were performed: serum neutralisation, haemagglutination inhibition, the enzyme-linked immunosorbent assay (ELISA), single radial haemolysis and immunofluorescence. For the evaluation of the tests, the results of serum neutralisation were used as a standard and compared with those of the other four methods. Immunofluorescence proved to be the most reliable method, followed by ELISA and haemagglutination inhibition, whereas single radial haemolysis showed the most deviations from seroneutralisation.

Zusammenfassung Mit der Checkerboard-Agar-Dilutionsmethode wurde die Kombinationswirkung von Cefotaxim mit Netilmicin und anderen Aminoglykosiden gegen 57 nicht fermentierende gramnegative Bakterien (Pseudomonas aeruginosa, Pseudomonas cepacia, Pseudomonas maltophilia, Pseudomonas fluorescensputida, Acinetobacter anitratus, Acinetobacter lwoffi) überprüft, die von Patienten mit im Krankenhaus erworbenen Infektionen isoliert worden waren. Durchschnittlich 39% der Nonfermenter wurden durch Cefotaxim-Aminoglykosid-Kombinationen additiv, 14% synergistisch gehemmt. Die additive und synergistische Wirkung war am besten beiP. aeruginosa, P. maltophilia undP. fluorescens-putida Stämmen, am niedrigsten gegenAcinetobacter Spezies; gegenP. cepacia Stämme trat kein Synergismus auf. Die additiven und/oder synergistischen Kombinationen senkten die minimalen Hemmkonzentrationen von Cefotaxim, Netilmicin und anderen Aminoglykosiden bei fast allen Stämmen auf therapeutisch erreichbare Serumkonzentrationen.

Forty-seven children and adolescents with brain tumors were examined by magnetic resonance imaging (MRI) after tumor resection. The typical changes and complications after surgery and chemotherapy, as well as the corresponding MRI findings, are discussed. Typical examples of boundary-layer lesions, tumor recurrences, hydrocephalus, porencephalic cysts, and hygromas are given.

The spinal cord in 56 children and adolescents was examined by magnetic resonance imaging (MRI) many years after neonatal surgery on a meningomyelocele (average age 12 years). In a high percentage of cases, the diagnosis “tethered cord” was made. Associated anomalies were found with a frequency of 21%. Typical findings are presented and the impact of these results on therapy planning is discussed.

An ecdysteroid UDP-glucosyltransferase (egt) gene was identified from the single (S) nucleocapsid nucleopolyhedrovirus of Heliothis armigera (HearNPV). In baculovirus-infected insects the viral enzyme (EGT) plays a pivotal role in abrogating the insect molting process. The open reading frame of the egt gene is 1545 nucleotides long, encoding a putative protein of 515 amino acids with a Mrof 59.1. The 5′-noncoding region contains a putative early (CAGT) and late (TAAG) motif for transcription initiation, a transcription enhancer sequence (CGTCGC) and two TATA boxes. A putative polyA signal, AATAAA, was found downstream of the translation stop codon. A putative signal peptide of 21 residues was present at the N-terminus of the EGT. The HearNPV egt gene has a high degree of nucleotide and amino acid sequence homology to the egt genes of Buzura suppressaria SNPV and Spodoptera exigua MNPV. The HearNPV EGT shares ten conserved motifs with other EGTs. A phylogenetic tree of twelve baculovirus EGTs was constructed by using maximum parsimony analysis, suggesting that SNPVs do not form a separate clade within the baculovirus family.

Full-length, truncated, and mature forms of the CryIA(b) insecticidal crystal protein gene of Bacillus thuringiensis were engineered into the p10 locus of Autographa californica nuclear polyhedrosis virus (AcNPV). A signal sequence of Heliothis virescens juvenile hormone esterase was introduced at the N-terminus of these constructs to induce secretion. All recombinants, except those containing the mature toxin, produced high levels of CryIA(b) ICPs in insect cells. Thirty percent of the intracellular protoxin was N-glycosylated, suggesting that the protoxin was translocated across the ER membrane. Secretion into the medium, however, was limited. The production of the mature toxin was poor as a result of its cytotoxicity to insect cells. In a bioassay against second instar Spodoptera exigua larvae, using a recombinant expressing the Androctonus australis scorpion toxin gene in the same p10 locus as a positive control, the median survival time of AcNPV recombinants expressing the various B. thuringiensis CryIA(b) ICP constructs was not significant different from that of wild-type AcNPV. This suggests that production and/or secretion of B. thuringiensis (pro)toxins by AcNPV p10 recombinant viruses does not increase insecticidal activity since (i) the protoxins produced are inactive and not likely to be activated in vivo; (ii) secretion of the B. thuringiensis protoxins is poor; and (iii) production of the mature toxins results in cytotoxicity. Copyright 1995, 1999 Academic Press

Summary In the present vaccination trial, 202 seronegative schoolchildren comprising both sexes and aged 11 to 12 years were vaccinated i.m. in the upper arm with either the subunit vaccine at a dosage of 600 CCA or 200 CCA or with a whole-virus vaccine at a dosage of 200 CCA, using the double-blind procedure. Both vaccines were prepared from the strain A/New Jersey/76 (x 5 3a-recombinant). The vaccination was followed four weeks later by a booster injection. In tests of local and systemic reactogenicity, it was found that at both dosages the subunit vaccine caused a low frequency of minor adverse reactions. The whole-virus vaccine was marked by a significantly higher rate of adverse reactions, whether of the local or systemic variety. The whole-virus vaccine had, however, a higher immunogenicity than the subunit vaccine, and due to the relatively high rate of adverse reactions it causes, it is not recommended for the vaccination of seronegative children. Because of its low reactogenicity, the subunit vaccine can be given at higher dosage, and it is a matter for consideration whether a better antibody response might not result from two booster injections.

Aphid transmission factor (ATF) activity of cauliflower mosaic virus (CaMV) gene II product was recovered after expression of the gene by a baculovirus recombinant. The expression product, when first acquired by aphids through parafilm membrane, was able to mediate the transmission of two aphid nontransmissible isolates (CM1841, CM14-184) providing the first direct evidence that the product of the gene II is the CaMV ATF. The CaMV ATF in its active conformation has a strong tendency to aggregate and all attempts at solubilizing it resulted in the loss of the ATF activity. The CaMV ATF was also expressed in Escherichia coli, using the pGEX 3X plasmid vector, as a fusion protein to glutathione S-transferase (GST) and was purified. The fusion product (GST-P18), whether purified or not, was not able to complement the transmission of transmission-defective isolates. However, when GST-P18 was added to some extracts from a plant infected with an aphid-transmissible isolate (Cabb B-JI), the transmission was inhibited. This suggests that it could be possible to block the in vitro transmission of CaMV using a molecule analogous to the ATF. Copyright 1993, 1999 Academic Press

Three functional domains in baculovirus p10 proteins have been postulated for aggregation, nuclear disintegration, and fibrillar structure formation (Van Oers et al., J. Gen. Virol. 74, 563-574, 1993). To study the specificity of these functions, a recombinant Autographa californica nuclear polyhedrosis virus (AcCR1) was constructed in which the coding sequence of the p10 gene was replaced with the p10 sequence of the distantly related Spodoptera exigua (Se) MNPV. In AcCR1-infected cells the SeMNPV p10 protein was produced at similarly high levels as AcMNPV p10 in wild type (wt) AcMNPV infections. Formation of fibrillar structures occurred in a similar fashion in SeMNPV and AcCR1-infected cells. Hence, the SeMNPV p10 protein retained the ability to associate into fibrillar structures when expressed in an otherwise AcMNPV context. Mixed infection with wt AcMNPV and AcCR1 indicated that only p10 proteins of the same species aggregate and that these aggregates associate into fibrillar structures. In contrast to S. exigua cells infected with AcMNPV or SeMNPV, S. exigua cells infected with AcCR1 failed to release polyhedra. This result indicated that interaction of p10 with at least one virus-specific factor is required for nuclear disintegration. Copyright 1994, 1999 Academic Press

Nasopharyngeal aspirates and nasopharyngeal swabs from 177 children exhibiting mild to severe clinical symptoms of whooping cough were tested by the polymerase chain reaction (PCR) and culture for the presence ofBordetella pertussis. In the PCR analysis amplifications of samples prepared with and without DNA extraction were compared. In 26 % of samples prepared without DNA extraction, the PCR was found to be inhibited, whereas no inhibition was detected after DNA extraction. Twelve percent (21/177) of the samples were positive in both culture and the PCR, and an additional 49 % (87/177) of the samples were positive exclusively in the PCR. Thirty-eight percent (8/21) of culture-positive patients and 63 % (55/87) of the patients in whom infection was detected only by PCR had mild or atypical clinical symptoms. Of these groups 26% (5/19) and 50 % (39/78), respectively, had been fully vaccinated with three or more doses of diphtheria, tetanus and pertussis vaccine.

Abstract.: We characterized an insertion mutant of the baculovirus Cydia pomonella granulovirus (CpGV), which contained a transposable element of 3.2 kb. This transposon, termed TCp3.2, has unusually long inverted terminal repeats (ITRs) of 756 bp and encodes a defective gene for a putative transposase. Amino acid sequence comparison of the defective transposase gene revealed a distant relationship to a putative transposon in Caenorhabditis elegans which also shares some similarity of the ITRs. Maximum parsimony analysis of the predicted amino acid sequences of Tc1- and mariner-like transposases available from the GenBank data base grouped TCp3.2 within the superfamily of Tc1-like transposons. DNA hybridization indicated that TCp3.2 originated from the genome of Cydia pomonella, which is the natural host of CpGV, and is present in less than 10 copies in the C. pomonella genome. The transposon TCp3.2 most likely was inserted into the viral genome during infection of host larvae. TCp3.2 and the recently characterized Tc1-like transposon TC14.7 (Jehle et al. 1995), which was also found in a CpGV mutant, represent a new family of transposons found in baculovirus genomes. The occasional horizontal escape of different types of host transposons into baculovirus genomes evokes the question about the possible role of baculoviruses as an interspecies vector in the horizontal transmission of insect transposons.

A solid phase radioimmunoassay on microtiter plates was adapted for the estimation of mumps antibodies using commercially available complement-fixing mumps antigen. The sensitivity of the test is superior to that of hemagglutination inhibition and lies in the same range as the virus neutralization test performed on chick fibroblasts. The method is useful for screening large series of human sera, for instance in connection with vaccination programs.