Gene-expression profiling of White spot syndrome virus in vivo

White spot syndrome virus, type species of the genus Whispovirus in the family Nimaviridae, is a large, double-stranded DNA (dsDNA) virus that infects crustaceans. The genome of the completely sequenced isolate WSSV-TH encodes 184 putative open reading frames (ORFs), the functions of which are largely unknown. To study the transcription of these ORFs, a DNA microarray was constructed, containing probes corresponding to nearly all putative WSSV-TH ORFs. Transcripts of 79 % of these ORFs could be detected in the gills of WSSV-infected shrimp (Penaeus monodon). Clustering of the transcription profiles of the individual genes during infection showed two major classes of genes: the first class reached maximal expression at 20 h post-infection (p.i.) (putative early) and the other class at 2 days p.i. (putative late). Nearly all major and minor structural virion-protein genes clustered in the latter group. These data provide evidence that, similar to other large, dsDNA viruses, the WSSV genes at large are expressed in a coordinated and cascaded fashion. Furthermore, the transcriptomes of the WSSV isolates WSSV-TH and TH-96-II, which have differential virulence, were compared at 2 days p.i. The TH-96-II genome encodes 10 ORFs that are not present in WSSV-TH, of which at least seven were expressed in P. monodon as well as in crayfish (Astacus leptodactylus), suggesting a functional but not essential role for these genes during infection. Expression levels of most other ORFs shared by both isolates were similar. Evaluation of transcription profiles by using a genome-wide approach provides a better understanding of WSSV transcription regulation and a new tool to study WSSV gene functio

Stable recombinant alpaca antibodies for detection of Tulip virus X

For detection of the plant pathogenic Tulip virus X (TuVX), a panel of six recombinant antibodies was identified. To this end, a repertoire of variable domains from heavy-chain immunoglobulins (VHH) was cloned from an alpaca, which had been immunized with TuVX. Binding domains were selected by phage display and panning on immobilized TuVX particles. Recombinant VHH antibodies were tested for sensitivity in a sandwich ELISA, and were demonstrated to be readily able to distinguish TuVX-infected tulip leaf material from uninfected leafs. No cross-reactivity of the VHH antibodies to related flexiviridae was observed. Recombinant VHHs maintained their reactivity upon storage at ¿20°C for over a year. The effect of incubation at higher temperatures for prolonged time was studied. Two out of three VHH proteins retained activity after several weeks of storage at 37°

Gene-expression profiling of White spot syndrome virus in vivo

White spot syndrome virus, type species of the genus Whispovirus in the family Nimaviridae, is a large, double-stranded DNA (dsDNA) virus that infects crustaceans. The genome of the completely sequenced isolate WSSV-TH encodes 184 putative open reading frames (ORFs), the functions of which are largely unknown. To study the transcription of these ORFs, a DNA microarray was constructed, containing probes corresponding to nearly all putative WSSV-TH ORFs. Transcripts of 79 % of these ORFs could be detected in the gills of WSSV-infected shrimp (Penaeus monodon). Clustering of the transcription profiles of the individual genes during infection showed two major classes of genes: the first class reached maximal expression at 20 h post-infection (p.i.) (putative early) and the other class at 2 days p.i. (putative late). Nearly all major and minor structural virion-protein genes clustered in the latter group. These data provide evidence that, similar to other large, dsDNA viruses, the WSSV genes at large are expressed in a coordinated and cascaded fashion. Furthermore, the transcriptomes of the WSSV isolates WSSV-TH and TH-96-II, which have differential virulence, were compared at 2 days p.i. The TH-96-II genome encodes 10 ORFs that are not present in WSSV-TH, of which at least seven were expressed in P. monodon as well as in crayfish (Astacus leptodactylus), suggesting a functional but not essential role for these genes during infection. Expression levels of most other ORFs shared by both isolates were similar. Evaluation of transcription profiles by using a genome-wide approach provides a better understanding of WSSV transcription regulation and a new tool to study WSSV gene functio

Generation of recombinant alpaca VHH antibody fragments for the detection of the mycotoxin ochratoxin A

To develop sensor technologies based on genetically engineered recognition elements, recombinant antibodies characterised by high stability are a prerequisite. Here we describe the first successful isolation of recombinant alpaca single-domain antibody fragments with high affinity to the mycotoxin ochratoxin A (OTA). Variable domains (VHH) of heavy-chain antibodies binding to OTA were cloned from an immunised alpaca. Selected VHH clones clustered in four major groups, based on protein sequence similarity. Six representative VHH antibody fragments were produced in Escherichia coli and characterised for their sensitivity and specificity by indirect competitive enzyme-linked immunosorbent assays. OTA concentrations causing 50% inhibition (IC50) ranged from 12 ng/ml for antibody fragment OCH-62 to 442 ng/ml for antibody fragment OCH-40. The IC50 of OCH-62 for ochratoxin B, an OTA analogue, was 476 ng/ml. No significant cross-reactivity was observed with common food constituents with structural similarities with parts of OTA, such as L-phenylalanine and coumarin. The performance of OCH-62 for detection of OTA in food matrices was comparable to the performance in buffer (white wine: IC50=17 ng/ml; red wine: IC50=21 ng/ml; instant coffee: IC50=17 ng/ml). In a membrane-based flow-through immunoassay, used for fast visual screening of samples, OCH-62 showed a cut-off level of 10 ng/ml OTA. Thereby, OCH-62 ranked among the best recombinant antibody fragments described for mycotoxins and is an excellent candidate for the design of food contamination sensors based on nanotechnology

Santalene synthase

The invention is directed to a santalene synthase, to a nucleic acid encoding said santalene synthase, to an expression vector comprising said nucleic acid, to a host cell comprising said expression vector, to a method of preparing santalene, to a method of preparing santalol and to a method of preparing a santalene synthase. The invention is further directed to an antibody specific for the santalane synthase

Generation of recombinant alpaca VHH antibody fragments for the detection of the mycotoxin ochratoxin A

To develop sensor technologies based on genetically engineered recognition elements, recombinant antibodies characterised by high stability are a prerequisite. Here we describe the first successful isolation of recombinant alpaca single-domain antibody fragments with high affinity to the mycotoxin ochratoxin A (OTA). Variable domains (VHH) of heavy-chain antibodies binding to OTA were cloned from an immunised alpaca. Selected VHH clones clustered in four major groups, based on protein sequence similarity. Six representative VHH antibody fragments were produced in Escherichia coli and characterised for their sensitivity and specificity by indirect competitive enzyme-linked immunosorbent assays. OTA concentrations causing 50% inhibition (IC50) ranged from 12 ng/ml for antibody fragment OCH-62 to 442 ng/ml for antibody fragment OCH-40. The IC50 of OCH-62 for ochratoxin B, an OTA analogue, was 476 ng/ml. No significant cross-reactivity was observed with common food constituents with structural similarities with parts of OTA, such as L-phenylalanine and coumarin. The performance of OCH-62 for detection of OTA in food matrices was comparable to the performance in buffer (white wine: IC50=17 ng/ml; red wine: IC50=21 ng/ml; instant coffee: IC50=17 ng/ml). In a membrane-based flow-through immunoassay, used for fast visual screening of samples, OCH-62 showed a cut-off level of 10 ng/ml OTA. Thereby, OCH-62 ranked among the best recombinant antibody fragments described for mycotoxins and is an excellent candidate for the design of food contamination sensors based on nanotechnology

A chicory cytochrome P450 mono-oxygenase CYP71AV8 for the oxidation of (+)-valencene

Chicory (Cichorium intybus L.), which is known to have a variety of terpene-hydroxylating activities, was screened for a P450 mono-oxygenase to convert (+)-valencene to (+)-nootkatone. A novel P450 cDNA was identified in a chicory root EST library. Co-expression of the enzyme with a valencene synthase in yeast, led to formation of trans-nootkatol, cis-nootkatol and (+)-nootkatone. The novel enzyme was also found to catalyse a three step conversion of germacrene A to germacra-1(10),4,11(13)-trien-12-oic acid, indicating its involvement in chicory sesquiterpene lactone biosynthesis. Likewise, amorpha-4,11-diene was converted to artemisinic acid. Surprisingly, the chicory P450 has a different regio-specificity on (+)-valencene compared to germacrene A and amorpha-4,11-dien

Santalene synthase

The invention is directed to a santalene synthase, to a nucleic acid encoding said santalene synthase, to an expression vector comprising said nucleic acid, to a host cell comprising said expression vector, to a method of preparing santalene, to a method of preparing santalol and to a method of preparing a santalene synthase. The invention is further directed to an antibody specific for the santalane synthase