Circulating tumor DNA analysis of the phase III VOYAGER trial: KIT mutational landscape and outcomes in patients with advanced gastrointestinal stromal tumor treated with avapritinib or regorafenib.

BACKGROUND: The current treatment paradigm of imatinib-resistant metastatic gastrointestinal stromal tumor (GIST) does not incorporate KIT/PDGFRA genotypes in therapeutic drug sequencing, except for PDGFRA exon 18-mutant GIST that is indicated for avapritinib treatment. Here, circulating tumor DNA (ctDNA) sequencing was used to analyze plasma samples prospectively collected in the phase III VOYAGER trial to understand how the KIT/PDGFRA mutational landscape contributes to tyrosine kinase inhibitor (TKI) resistance and to determine its clinical validity and utility. PATIENTS AND METHODS: VOYAGER (N = 476) compared avapritinib with regorafenib in patients with KIT/PDGFRA-mutant GIST previously treated with imatinib and one or two additional TKIs (NCT03465722). KIT/PDGFRA ctDNA mutation profiling of plasma samples at baseline and end of treatment was assessed with 74-gene Guardant360® CDx. Molecular subgroups were determined and correlated with outcomes. RESULTS: A total of 386/476 patients with KIT/PDGFRA-mutant tumors underwent baseline (pre-trial treatment) ctDNA analysis; 196 received avapritinib and 190 received regorafenib. KIT and PDGFRA mutations were detected in 75.1% and 5.4%, respectively. KIT resistance mutations were found in the activation loop (A-loop; 80.4%) and ATP-binding pocket (ATP-BP; 40.8%); 23.4% had both. An average of 2.6 KIT mutations were detected per patient; 17.2% showed 4-14 different KIT resistance mutations. Of all pathogenic KIT variants, 28.0% were novel, including alterations in exons/codons previously unreported. PDGFRA mutations showed similar patterns. ctDNA-detected KIT ATP-BP mutations negatively prognosticated avapritinib activity, with a median progression-free survival (mPFS) of 1.9 versus 5.6 months for regorafenib. mPFS for regorafenib did not vary regardless of the presence or absence of ATP-BP/A-loop mutants and was greater than mPFS with avapritinib in this population. Secondary KIT ATP-BP pocket mutation variants, particularly V654A, were enriched upon disease progression with avapritinib. CONCLUSIONS: ctDNA sequencing efficiently detects KIT/PDGFRA mutations and prognosticates outcomes in patients with TKI-resistant GIST treated with avapritinib. ctDNA analysis can be used to monitor disease progression and provide more personalized treatment.

Quality standards and guidelines for test validation for infectious diseases in veterinary laboratories.

The World Organisation for Animal Health Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, Chapter 1.1.1. summarises the most relevant governance and managerial aspects of veterinary testing laboratories, and Chapter 1.1.5. introduces quality management. Both chapters are based on the International Organization for Standardization/International Electrotechnical Commission standard, ISO/IEC 17025:2005 'General requirements for the competence of testing and calibration laboratories'. This paper provides an update of standards and regulatory bodies relevant for accreditation of quality management systems (QMS), with a focus on ISO/IEC 17025:2017 for testing and calibration laboratories. Important issues and considerations that a laboratory should address in the design and maintenance of its QMS are highlighted and examples provided, in particular aspects of test validation and verification, including measurement uncertainty (MU). A QMS aims to address all aspects of the laboratory operation, including staff, organisational structure, processes, and procedures. Accreditation of a diagnostic laboratory requires three notable components: (a) independent or third-party assessment; (b) suitably validated tests performed by proficient laboratory operators in an adequately equipped laboratory; and (c) ongoing internal and external quality control. Together, these components ensure a test outcome is the result of a standardised process and structured peer review, and demonstrate both competency and ability to produce technically valid diagnostic results that will meet the needs of customers - veterinarians, animal owners, regulators, organisations and industry - as well as the needs of decision-makers involved in animal health and surveillance programmes.

Le chapitre 1.1.1 du Manuel des tests de diagnostic et des vaccins pour les animaux terrestres de l'Organisation mondiale pour la santé animale donne une vue d'ensemble des principaux aspects de la gouvernance et de la gestion d'un laboratoire de diagnostic vétérinaire tandis que le chapitre 1.1.5 introduit aux principes de la gestion de la qualité dans les laboratoires. Les deux chapitres reposent sur la norme de l'Organisation internationale de normalisation/Commission électrotechnique internationale ISO/IEC 17025:2005, « Exigences générales concernant la compétence des laboratoires d'étalonnages et d'essais ¼. Les auteurs font le point sur l'état actuel des normes et des organismes de réglementation pertinents en matière d'accréditation des systèmes de gestion de la qualité , en mettant l'accent sur la norme ISO/IEC 17025:2017 qui est plus précisément axée sur les laboratoires d'essais et d'étalonnage. Les auteurs mettent en avant un certain nombre de questions et de considérations importantes qu'un laboratoire devrait prendre en compte lors de la conception et de la mise en œuvre continue de son système de gestion de la qualité et les illustrent d'exemples relatifs à des aspects particuliers de la validation et du contrôle des performances d'un test, notamment l'incertitude des mesures. Un système de gestion de la qualité doit courir tous les aspects opérationnels d'un laboratoire, y compris le personnel, la structure organisationnelle, les processus et les procédures. L'accréditation d'un laboratoire de diagnostic repose sur trois composantes majeures : a) l'évaluation, qui doit être conduite de manière indépendante ou par des tiers ; b) des tests validés de manière appropriée et utilisés par des opérateurs de laboratoire qualifiés dans un laboratoire correctement équipé; c) un contrôle de la qualité continu, à la fois interne et externe. Prises ensemble, ces composantes garantissent que les résultats d'un test sont le fruit d'un processus normalisé et d'un examen structuré et conduit par des pairs, et démontrent aussi bien la compétence que la capacité à produire des résultats diagnostiques robustes sur le plan technique et répondant aux besoins des clients ­ vétérinaires, propriétaires d'animaux, régulateurs, organisations et secteur privé ­ ainsi qu'aux besoins des décideurs en charge des programmes de santé animale et de surveillance.

En el capítulo 1.1.1 del Manual de las Pruebas de Diagnóstico y de las Vacunas para los Animales Terrestres de la Organización Mundial de Sanidad Animal (OIE) se resumen los aspectos más importantes de la dirección y la gestión de laboratorios de análisis veterinarios, mientras que en el capítulo 1.1.5 se aborda el tema de la gestión de la calidad. Ambos capítulos están basados en la norma ISO/IEC 17025:2005, "Requisitos generales para la competencia de los laboratorios de ensayo y de calibración" de la Organización Internacional de Normalización y la Comisión Electrotécnica Internacional. Los autores ofrecen información actualizada sobre las normas aplicables y los organismos de reglamentación competentes por lo que respecta a la certificación de sistemas de gestión de la calidad, partiendo básicamente de las disposiciones de la norma ISO/IEC 17025:2017 que se aplican a los laboratorios de ensayo y de calibración. También destacan las cuestiones y consideraciones importantes que un laboratorio debe tener en cuenta para concebir y mantener su sistema de gestión de la calidad, en particular los aspectos relativos a la validación y verificación de pruebas, incluida la incertidumbre de medición, y ofrecen ejemplos al respecto. Un sistema de gestión de la calidad tiene por objetivo cubrir todos los aspectos del funcionamiento de un laboratorio, lo que comprende su personal, su estructura organizativa y sus procesos y protocolos. La acreditación de un laboratorio de diagnóstico consta de tres componentes fundamentales: a) una evaluación por parte de un tercero independiente; b) la realización de pruebas debidamente validadas, a cargo de técnicos de laboratorio competentes, en un laboratorio convenientemente equipado; y c) controles continuos de la calidad, tanto internos como externos. La suma de estos componentes garantiza que los resultados de un ensayo sean fruto de un proceso normalizado y de una revisión por homólogos estructurada y demuestra que el laboratorio posee tanto la competencia como la capacidad necesarias para obtener resultados de diagnóstico técnicamente válidos, que respondan a las necesidades tanto de los clientes (veterinarios, propietarios de animales, organismos de reglamentación, organizaciones y entidades industriales) como de las instancias decisorias que intervienen en los programas de sanidad animal y vigilancia zoosanitaria.

Your assay has changed - is it still 'fit for purpose'? What evaluation is required.

A reliable laboratory assay is an essential tool for the diagnosis or surveillance of most animal diseases. Before routine use, assays should be appropriately validated to ensure that they have performance characteristics that provide reliable results and can be used for the intended purpose. It is inevitable that, over time, changes will need to be made to assay reagents, to the assay format, to test a different species or for implementation in a new laboratory. Whenever there is a change (whether it be components, application or location), it is essential to establish whether the new circumstances affect the biological basis and properties of the assay. If the modifications do not affect the biological basis of the assay, the changes might be considered minor and a verification study can be conducted to confirm that the performance characteristics have not been adversely affected. Major changes require a new validation to be carried out. A method comparability study, where original and modified assays are run concurrently to test the same sample panel, provides an extremely robust comparison. However, comparability studies are not always an option, especially for the introduction of a method to a new laboratory. Access to original validation data and suitable reference sample panels then becomes essential to provide evidence that the assay remains 'fit for the intended purpose'.

Les essais de laboratoire fiables constituent des outils essentiels pour le diagnostic ou la surveillance d'une majorité de maladies animales. Avant d'être utilisés en routine, les essais doivent faire l'objet d'une validation appropriée, destinée à s'assurer qu'ils possèdent les caractéristiques de performance nécessaires pour générer des résultats fiables et à déterminer qu'ils peuvent être utilisés pour la finalité prévue. Au fil du temps, il est inévitable que certains changements soient apportés aux réactifs de l'essai ou à son format, visant par exemple à appliquer le test sur autre espèce animale ou dans un nouveau laboratoire. À chaque changement introduit (qu'il s'agisse d'une composante du test, de sa modalité d'application ou du lieu où il est conduit), il est essentiel de déterminer si ces nouvelles circonstances affectent la base biologique et les propriétés de l'essai. Si les modifications n'affectent pas la base biologique de l'essai, les changements peuvent être considérés comme mineurs et une étude de contrôle des performances pourra être réalisée pour confirmer que les caractéristiques de performance n'ont pas subi d'altération indésirable. En cas de changement majeur, une nouvelle étude de validation devra être réalisée. L'étude de comparabilité de méthodes, qui consiste à réaliser simultanément l'essai original et l'essai modifié sur un même panel d'échantillons fournit une comparaison extrêmement robuste. Néanmoins, dans certaines situations les études de comparabilité ne sont pas une option, notamment lorsqu'il s'agit d'introduire la méthode modifiée dans un nouveau laboratoire. Il devient alors indispensable de pouvoir accéder aux données de validation originales et de disposer de panels d'échantillons de référence appropriés afin de fournir la preuve que l'essai est toujours « apte à l'emploi ¼ qui lui a été assigné.

Un ensayo de laboratorio fiable es una herramienta básica para el diagnóstico o la vigilancia de la mayoría de las enfermedades animales. Antes de poder emplear de forma sistemática un ensayo es preciso validarlo debidamente, para tener la seguridad de que presente características de rendimiento adecuadas, que deparen resultados fiables, y de que se ajuste al propósito previsto. Con el tiempo, inevitablemente, será preciso modificar los reactivos y el formato del ensayo, con objeto de aplicarlo a una especie diferente o de practicarlo en un nuevo laboratorio. Siempre que haya un cambio en el ensayo (ya sea en sus componentes o en su modo o lugar de aplicación), será esencial determinar si las nuevas circunstancias influyen en su base biológica o en sus propiedades. Cuando las modificaciones no incidan en su base biológica, se podrá considerar que los cambios son de importancia menor y se podrá realizar un estudio de verificación para comprobar que las características de rendimiento no se han visto negativamente afectadas. Los cambios de mayor entidad exigen un nuevo proceso de validación. Los estudios de comparación de métodos, en los que paralelamente se aplican la técnica original y la modificada a un mismo panel de muestras, deparan una comparación sumamente robusta. Sin embargo, los estudios de comparabilidad no siempre son una posibilidad factible, sobre todo cuando se trata de empezar a aplicar un método en un nuevo laboratorio. En tales casos resultará fundamental tener acceso a los datos de validación originales y a paneles adecuados de muestras de referencia para asegurarse de que el ensayo siga siendo 'idóneo para el propósito previsto'.

Oral lenalidomide with rituximab in relapsed or refractory diffuse large cell, follicular and transformed lymphoma: a phase II clinical trial.

Lenalidomide-rituximab therapy is effective in grade 1-2 follicular and mantle cell lymphoma, but its efficacy in diffuse large B-cell lymphoma (DLBCL), transformed large cell lymphoma (TL) and grade 3 follicular lymphoma (FLG3) is unknown. In this phase II trial, 45 patients with relapsed or refractory DLBCL (n=32), TL (n=9) or FLG3 (n=4) who had received 1-4 prior lines of treatment were given 20 mg oral lenalidomide on days 1-21 of each 28-day cycle, and intravenous rituximab (375 mg/m(2)) weekly during cycle 1. Grade 3/4 hematological toxicities included neutropenia (53%), lymphopenia (40%), thrombocytopenia (33%), leukopenia (27%) and anemia (18%), with a median follow-up time of 29.1 months (range 14.7-52.0 months). Overall response (OR) rate was 33%; median response duration was 10.2 months. Median progression-free survival (PFS) and overall survival (OS) were 3.7 and 10.7 months, respectively. Nine of the 15 responding patients (three partial response (PR), six complete response (CR)) proceeded with stem cell transplantation (SCT) and were censored at the time of transplantation. When data were analyzed without censoring, median PFS remained 3.7 months and response duration increased to 30.9 months. Rituximab plus oral lenalidomide is well tolerated and effective for patients with relapsed/refractory DLBCL and TL. SCT after lenalidomide-rituximab is associated with prolonged response duration.

Epizootics of sudden death in tammar wallabies (Macropus eugenii) associated with an orbivirus infection.

Epizootics of sudden death in tammar wallabies (Macropus eugenii) occurred at six research facilities and zoological gardens in New South Wales, Australia, in late 1998 and at one Queensland research facility in March 1999. There were 120 confirmed tammar wallaby deaths during this period; however, population censuses indicated that up to 230 tammar wallabies may have died. The majority of animals died without premonitory signs. A small proportion of wallabies exhibited increased respiratory rate, sat with a lowered head shortly before death or were discovered in lateral recumbency, moribund and with muscle fasciculations. Gross postmortem findings consistently included massive pulmonary congestion, mottled hepatic parenchyma and subcutaneous oedema throughout the hindlimbs and inguinal region. Approximately 30% of the animals examined also had extensive haemorrhage within the fascial planes and skeletal muscle of the hindlimb adductors, inguinal region, ventral thorax, dorsal cervical region and perirenal retroperitoneal area. The tissues of affected animals became autolytic within a short period after death. Bacteriological examination of tissues from 14 animals did not provide any significant findings. Toxicological examination of the gastric and colonic contents of four animals did not reveal evidence of brodifacoume or other rodenticides. Viruses from the Eubenangee serogroup of the Orbivirus genus were isolated from the cerebral cortex of nine, and the myocardium of two, tammar wallabies and the liver and intestine of another tammar wallaby. A similar orbivirus was also isolated from the cerebrospinal fluid of another tammar wallaby that died suddenly. The disease agent appears to be a previously unrecognised orbivirus in the Eubenangee serogroup. This is the first report of epizootics of sudden deaths in tammar wallabies apparently associated with an orbivirus infection.

Cultured skin fibroblast cells derived from bluetongue virus-inoculated sheep and field-infected cattle are not a source of late and protracted recoverable virus.

A recent hypothesis to explain the recurrence of bluetongue disease after winter seasonal absences of the vector has suggested a role for persistent infection of sheep. This report presents combined independent work from two laboratories investigating the possible recovery of Bluetongue virus (BTV) over a protracted period after infection of both sheep and cattle. Prior to infection with either cell-culture-adapted or non-culture-adapted BTV, sheep were subjected to a preliminary exposure to Culicoides sp. insects, which reportedly facilitates recovery of virus from infected sheep several months post-infection (p.i.). A series of skin biopsies at different intervals p.i. was used to establish skin fibroblast (SF) cultures from which attempts were made to detect virus by isolation and by molecular and immunological methods. Also examined was the effect on virus recovery of additional exposure to Culicoides sp. prior to skin biopsy during the post-inoculation period. A herd of cattle sentinels for surveillance of natural BTV infection in northern Australia was monitored prospectively for seroconversion. Evidence of infection initiated attempted virus recovery by establishing SF cultures. It was found that in both cattle and sheep there was not a protracted period over which BTV could be recovered from SF cultures. The data do not support a general hypothesis that BTV persists in either sheep or cattle.

Molecular diagnosis of lyssaviruses and sequence comparison of Australian bat lyssavirus samples.

OBJECTIVE: To evaluate and implement molecular diagnostic tests for the detection of lyssaviruses in Australia. DESIGN: A published hemi-nested reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of all lyssavirus genotypes was modified to a fully nested RT-PCR format and compared with the original assay. TaqMan assays for the detection of Australian bat lyssavirus (ABLV) were compared with both the nested and hemi-nested RT-PCR assays. The sequences of RT-PCR products were determined to assess sequence variations of the target region (nucleocapsid gene) in samples of ABLV originating from different regions. RESULTS: The nested RT-PCR assay was highly analytically specific, and at least as analytically sensitive as the hemi-nested assay. The TaqMan assays were highly analytically specific and more analytically sensitive than either RT-PCR assay, with a detection level of approximately 10 genome equivalents per microl. Sequence of the first 544 nucleotides of the nucleocapsid protein coding sequence was obtained from all samples of ABLV received at Australian Animal Health Laboratory during the study period. CONCLUSION: The nested RT-PCR provided a means for molecular diagnosis of all tested genotypes of lyssavirus including classical rabies virus and Australian bat lyssavirus. The published TaqMan assay proved to be superior to the RT-PCR assays for the detection of ABLV in terms of analytical sensitivity. The TaqMan assay would also be faster and cross contamination is less likely. Nucleotide sequence analyses of samples of ABLV from a wide geographical range in Australia demonstrated the conserved nature of this region of the genome and therefore the suitability of this region for molecular diagnosis.

Pathogenesis studies with Australian bat lyssavirus in grey-headed flying foxes (Pteropus poliocephalus).

OBJECTIVE: To examine the susceptibility of the grey-headed flying fox (Pteropus poliocephalus) to Australian bat lyssavirus (ABL), and to provide preliminary observations on the pathogenesis of the disease in flying foxes. PROCEDURE: Ten flying foxes were inoculated intramuscularly with ABL, and four with a bat-associated rabies virus. Inoculated animals were observed daily, and clinical samples collected every 9 to 14 days. Animals with abnormal clinical signs were euthanased, and samples collected for histological, serological, virological and immunohistological examinations. At 3 months post inoculation (PI), all survivors were euthanased, and each submitted to a similar examination. RESULTS: Three ABL-inoculated flying foxes, and two rabies-inoculated animals developed abnormal clinical signs between 15 and 24 days PI. All three ABL-inoculated animals had histological lesions consistent with a lyssavirus infection, and lyssaviral antigen was identified in the central nervous system (CNS) of each. Virus was isolated from the brain of two affected animals. Of the rabies-inoculated flying foxes, both had histological lesions and viral antigen in the CNS. Virus was recovered from the brain of only one. None of the five affected flying foxes developed anti-lyssavirus antibodies, but, by 3 months PI, five of the seven ABL-inoculated survivors, and one of the two rabies virus-inoculated survivors, had seroconverted. The dynamics of the immune responses were quite variable. CONCLUSIONS: The response of flying foxes to ABL, administered by a peripheral route of inoculation, was similar to that of bats inoculated peripherally with bat-derived rabies viruses.

Experimental infections of pigs with Japanese encephalitis virus and closely related Australian flaviviruses.

The flavivirus Japanese encephalitis (JE) virus has recently emerged in the Australasian region. To investigate the involvement of infections with related enzootic flaviviruses, namely Murray Valley encephalitis (MVE) virus and Kunjin (KUN) virus, on immunity of pigs to JE virus and to provide a basis for interpretation of serologic data, experimental infections were conducted with combinations of these viruses. Antibody responses to primary and secondary infections were evaluated using panels of monoclonal antibody-based blocking enzyme-linked immunosorbent assays and microtiter serum neutralization tests (mSNTs). Identification of the primary infecting virus was possible only using the mSNTs. Following challenge, unequivocal diagnosis was impossible due to variation in immune responses between animals and broadened and/or anamnestic responses. Viremia for JE virus was readily detected in pigs following primary infection, but was not detected following prior exposure to MVE or KUN viruses. Boosted levels of existing cross-neutralizing antibodies to JE virus suggested a role for this response in suppressing JE viremia.

Influence of transfer RNA tertiary structure on aminoacylation efficiency by glutaminyl and cysteinyl-tRNA synthetases.

The position of the tertiary Levitt pair between nucleotides 15 and 48 in the transfer RNA core region suggests a key role in stabilizing the joining of the two helical domains, and in maintaining the relative orientations of the D and variable loops. E. coli tRNA(Gln) possesses the canonical Pu15-Py48 trans pairing at this position (G15-C48), while the tRNA(Cys) species from this organism instead features an unusual G15-G48 pair. To explore the structural context dependence of a G15-G48 Levitt pair, a number of tRNA(Gln) species containing G15-G48 were constructed and evaluated as substrates for glutaminyl and cysteinyl-tRNA synthetases. The glutaminylation efficiencies of these mutant tRNAs are reduced by two to tenfold compared with native tRNA(Gln), consistent with previous findings that the tertiary core of this tRNA plays a role in GlnRS recognition. Introduction of tRNA(Cys) identity nucleotides at the acceptor and anticodon ends of tRNA(Gln) produced a tRNA substrate which was efficiently aminoacylated by CysRS, even though the tertiary core region of this species contains the tRNA(Gln) G15-C48 pair. Surprisingly, introduction of G15-G48 into the non-cognate tRNA(Gln) tertiary core then significantly impairs CysRS recognition. By contrast, previous work has shown that CysRS aminoacylates tRNA(Cys) core regions containing G15-G48 with much better efficiency than those with G15-C48. Therefore, tertiary nucleotides surrounding the Levitt pair must significantly modulate the efficiency of aminoacylation by CysRS. To explore the detailed nature of the structural interdependence, crystal structures of two tRNA(Gln) mutants containing G15-G48 were determined bound to GlnRS. These structures show that the larger purine ring of G48 is accommodated by rotation into the syn position, with the N7 nitrogen serving as hydrogen bond acceptor from several groups of G15. The G15-G48 conformations differ significantly compared to that observed in the native tRNA(Cys) structure bound to EF-Tu, further implicating an important role for surrounding nucleotides in maintaining the integrity of the tertiary core and its consequent ability to present crucial recognition determinants to aminoacyl-tRNA synthetases.

Crystallization and preliminary diffraction analysis of Escherichia coli cysteinyl-tRNA synthetase.

Crystals of the 52 kDa monomeric Escherichia coli cysteinyl-tRNA synthetase complexed with ATP and cysteine have been grown by hanging-drop vapor diffusion from solutions containing ammonium sulfate as the precipitating agent. The crystals form long hexagonal rods in the space group P321 with unit-cell dimensions a = b = 82.3, c = 168.9 A. There is one enzyme molecule in the asymmetric unit. A complete native data set has been collected from a rotating-anode source to a resolution of 2.7 A at 103 K, with an Rmerge of 6.7%.

Metal ion-mediated substrate-assisted catalysis in type II restriction endonucleases.

The 2.15-A resolution cocrystal structure of EcoRV endonuclease mutant T93A complexed with DNA and Ca2+ ions reveals two divalent metals bound in one of the active sites. One of these metals is ligated through an inner-sphere water molecule to the phosphate group located 3' to the scissile phosphate. A second inner-sphere water on this metal is positioned approximately in-line for attack on the scissile phosphate. This structure corroborates the observation that the pro-SP phosphoryl oxygen on the adjacent 3' phosphate cannot be modified without severe loss of catalytic efficiency. The structural equivalence of key groups, conserved in the active sites of EcoRV, EcoRI, PvuII, and BamHI endonucleases, suggests that ligation of a catalytic divalent metal ion to this phosphate may occur in many type II restriction enzymes. Together with previous cocrystal structures, these data allow construction of a detailed model for the pretransition state configuration in EcoRV. This model features three divalent metal ions per active site and invokes assistance in the bond-making step by a conserved lysine, which stabilizes the attacking hydroxide ion nucleophile.

Identification of a firefly luciferase active site peptide using a benzophenone-based photooxidation reagent.

Firefly luciferase catalyzes the highly efficient emission of yellow-green light from substrate luciferin by a series of reactions that require MgATP and molecular oxygen. We prepared 2-(4-benzoylphenyl)thiazole-4-carboxylic acid (BPTC), a novel benzophenone-based substrate analog, intending to use it in photoaffinity labeling studies to probe the luciferase active site. Instead, we found that while BPTC was a potent photoinactivating reagent for firefly luciferase, it was not a photoaffinity labeling agent. Using proteolysis, reverse phase high-performance liquid chromatography, tandem high performance liquid chromatography-electrospray ionization mass spectrometry, and Edman sequencing, we identified a single luciferase peptide, 244HHGF247, the degradation of which was directly correlated to luciferase photoinactivation. Results of enzyme kinetics and related studies were consistent with this peptide being at or near the luciferin binding site. Further, peptide model studies and additional investigations on the nature of the photoinactivation process strongly suggested that BPTC catalyzed the formation of singlet oxygen at the active site of the enzyme. We describe here an uncommon example of active site-directed photooxidation of an enzyme by singlet oxygen.

EHDV-1, a new Australian serotype of epizootic haemorrhagic disease virus isolated from sentinel cattle in the Northern Territory.

In 1992, a virus (DPP2209) isolated from sentinel cattle located at Coastal Plains Research Station, latitude 12 degrees 39'S, longitude 131 degrees 20'E, approximately 60 km east of Darwin, Northern Territory. This virus was identified as a serotype of epizootic haemorrhagic disease (EHD) of deer virus previously undescribed in Australia. An additional 17 isolation of this virus were made from eight animals during the period February to May. Electron microscopic studies showed the presence of orbivirus-like structures. Serogrouping ELISA, indirect immunofluorescence assay and the serogrouping plaque reduction neutralisation test indicated the virus was a member of the epizootic haemorrhagic disease serogroup. Serotype specific plaque reduction neutralisation tests, indicated the virus was a member of the epizootic haemorrhagic disease serogroup not previously isolated in Australia. Analysis of the VP3 gene confirmed this observation. Cross neutralisation testing of the isolate with known epizootic haemorrhagic disease serotype viruses including endemic Australian and exotic strains identified isolate DPP2209 as epizootic haemorrhagic disease virus serotype 1.

Detection by ELISA of bluetongue antigen directly in the blood of experimentally infected sheep.

An antigen-capture ELISA (Ag-ELISA) was developed to detect bluetongue virus (BTV) antigen directly from blood samples. Four blood preparations [whole blood, buffy coat, washed red blood cells (RBC) and plasma] taken pre-inoculation and on days 6 to 9 post-inoculation (PI) were used in the ELISA to study antigenaemia in forty sheep, each experimentally infected with one of 20 South African BTV serotypes. Seventeen of the 20 serotypes were detected and 27 of the 40 sheep were at some stage Ag-ELISA positive. Over the period of sampling, Ag-ELISA positive results were most frequently returned from whole blood taken on days 6 and 7 PI. However in some instances the quantity and/or duration of BTV antigenaemia was greater in buffy coat and washed RBC preparations. In a selection of samples examined, positive Ag-ELISA results were generally obtained when samples had an infectious virus titre in eggs of > 10(3.2) egg lethal doses (ELD50/ml). The appearance and duration of detectable antigenaemia was compared with the development of clinical signs and antibody responses of infected sheep. On days 6 and 7 PI the presence of fever (> 40 degrees C) was indicative to the likelihood of detectable antigenaemia. After day 5 PI antigenaemia declined and clinical signs of swollen face and inflamed feet appeared together with the first detectable antibody response. The Ag-ELISA, when used in conjunction with clinical observations and serologic data, should be useful as a rapid diagnostic procedure for bluetongue disease.

The use of an indirect ELISA with protein G-peroxidase conjugate and a blocking ELISA to demonstrate recent bluetongue infection in sheep.

The humoral immune response of sheep infected with bluetongue virus serotypes 3, 9 and 16 was monitored by plaque inhibition (PI), blocking ELISA (BELISA) and indirect ELISA over a period of 63 days post-infection. Results indicated that testing of a single plasma or serum sample by both a BELISA and an indirect ELISA using a recombinant streptococcal protein G (PrG) peroxidase conjugate enabled an assessment of the proximity of a recent infection based on the failure of PrG to bind ovine IgM class antibodies. When BELISA and indirect ELISA results were expressed as a ratio, values indicative of recent infection (> or = 5) were observed for an average duration of 16.5 days (range 8 to 23 days) following the initial detection of antibody by BELISA. This approach has potential to improve diagnosis of a wide range of virus infections by providing an indicator for the relationship of serological status with a recent infection. However, where reinfection may occur, as with bluetongue virus, alternative methods may be required for definitive diagnosis.

Identification of epizootic haemorrhagic disease of deer virus serotypes using a fluorescence inhibition test.

A fluorescence inhibition test (FIT) is described for serotyping rapidly isolates of epizootic haemorrhagic disease of deer virus (EHDV). The test used a serogroup-reactive monoclonal antibody in a immunofluorescence procedure to detect virus which resisted neutralisation by antisera to any of the eight known EHDV serotypes. The EHDV FIT provided an accurate serotype identification procedure for all eight reference serotypes and, in comparison with the plaque inhibition assay, abbreviated the serotyping process by three to four days.

The tropical bedbug Cimex hemipterus near the southernmost extent of its range.

The average degree of infestation of Cimex hemipterus in Zulu huts in South Africa is the same as that of C. lectularius. Nymphal stages of C. hemipterus can be distinguished by head-width measurements and numbers of lateral pronotal hairs.