Bluetongue Virus Infection of Goats: Re-Emerged European Serotype 8 vs. Two Atypical Serotypes.

In recent years, numerous atypical Bluetongue virus (BTV) strains have been discovered all around the world. Atypical BTV strains are phylogenetically distinct from the classical BTV serotypes 1-24 and differ in terms of several biological features. For the first time, the atypical strains BTV-25-GER2018 and BTV-33-MNG3/2016 as well as the re-emerged classical strain BTV-8-GER2018 were evaluated comparatively in a pathogenesis study in goats-the natural host of atypical BTV. A substantial number of in-contact animals were included in this study to detect potential contact transmissions of the virus. After infection, EDTA blood, ocular, nasal and oral swab samples as well as serum were collected regularly and were used for virological and serological analyses, respectively. Our study showed differences in the immunological reaction between the two atypical BTV strains (no group-specific antibody detection) and the classical BTV strain BTV-8-GER2018 (group-specific antibody detection). Furthermore, we observed an increase in the total WBC count (neutrophils and lymphocytes) in goats infected with the atypical BTV strains. No horizontal transmission was seen for all three strains. Our study suggests that the atypical BTVs used in the trial differ from classical BTVs in their immunopathogenesis. However, no evidence of direct contact transmission was found.

LAMP4yaws: Treponema pallidum, Haemophilus ducreyi loop mediated isothermal amplification - protocol for a cross-sectional, observational, diagnostic accuracy study.

INTRODUCTION: Yaws, caused by the bacterium Treponema pallidum subsp. pertenue, is a neglected tropical disease targeted for eradication by 2030. Improved diagnostics will be essential to meet this goal. Diagnosis of yaws has relied heavily on clinical and serological tools. However, the presence of coendemic cutaneous skin ulcer diseases, such as lesions caused by Haemophilus ducreyi (HD), means these techniques do not provide a reliable diagnosis. Thus, new diagnostic tools are needed. Molecular tools such as PCR are ideal, but often expensive as they require trained technicians and laboratory facilities, which are often not available to national yaws programmes. METHODS AND ANALYSIS: The LAMP4yaws project is a cross-sectional, observational, diagnostic accuracy study of a combined Treponema pallidum (TP) and HD loop mediated isothermal amplification (TPHD-LAMP) test performed under real world conditions in three endemic countries in West Africa. Individuals with serologically confirmed yaws will be recruited in Cameroon, Côte d'Ivoire and Ghana. Each participant will provide paired swabs, one of which will be sent to the respective national reference laboratory for yaws quantitative PCR and the other will be tested for both TP and HD using the TPHD-LAMP test at local district laboratories. Sensitivity and specificity of the TPHD-LAMP test will be calculated against the reference standard qPCR. We will also assess the acceptability, feasibility and cost-effectiveness of the test. We anticipate that results from this study will support the adoption of the TPHD-LAMP test for use in global yaws eradication efforts. ETHICS AND DISSEMINATION: We have received ethical approval from all relevant institutional and national ethical committees. All participants, or their parents or guardians, must provide written informed consent prior to study enrolment. Study results will be published in an open access journal and disseminated with partners and the World Health Organization. TRIAL REGISTRATION NUMBER: NCT04753788.

Putative Novel Atypical BTV Serotype '36' Identified in Small Ruminants in Switzerland.

We identified a putative novel atypical BTV serotype '36' in Swiss goat flocks. In the initial flock clinical signs consisting of multifocal purulent dermatitis, facial oedema and fever were observed. Following BTV detection by RT-qPCR, serotyping identified BTV-25 and also a putative novel BTV serotype in several of the affected goats. We successfully propagated the so-called "BTV-36-CH2019" strain in cell culture, developed a specific RT-qPCR targeting Segment 2, and generated the full genome by high-throughput sequencing. Furthermore, we experimentally infected goats with BTV-36-CH2019. Regularly, EDTA blood, serum and diverse swab samples were collected. Throughout the experiment, neither fever nor clinical disease was observed in any of the inoculated goats. Four goats developed BTV viremia, whereas one inoculated goat and the two contact animals remained negative. No viral RNA was detected in the swab samples collected from nose, mouth, eye, and rectum, and thus the experimental infection of goats using this novel BTV serotype delivered no indications for any clinical symptoms or vector-free virus transmission pathways. The subclinical infection of the four goats is in accordance with the reports for other atypical BTVs. However, the clinical signs of the initial goat flock did most likely not result from infection with the novel BTV-36-CH0219.

Isolation and Cultivation of a New Isolate of BTV-25 and Presumptive Evidence for a Potential Persistent Infection in Healthy Goats.

Recently, several so-called "atypical" Bluetongue virus (BTV) serotypes were discovered, including BTV-25 (Toggenburg virus), in Switzerland. Most "atypical" BTV were identified in small ruminants without clinical signs. In 2018, two goats from a holding in Germany tested positive for BTV-25 genome by RT-qPCR prior to export. After experimental inoculation of the two goats with the BTV-25 positive field blood samples for generation of reference materials, viremia could be observed in one animal. For the first time, the BTV-25-related virus was isolated in cell culture from EDTA-blood and the full genome of isolate "BTV-25-GER2018" could be generated. BTV-25-GER2018 was only incompletely neutralized by ELISA-positive sera. We could monitor the BTV-25 occurrence in the respective affected goat flock of approximately 120 goats over several years. EDTA blood samples were screened with RT-qPCR using a newly developed BTV-25 specific assay. For serological surveillance, serum samples were screened using a commercial cELISA. BTV-25-GER2018 was detected over 4.5 years in the goat flock with intermittent PCR-positivity in some animals, and with or without concomitantly detected antibodies since 2015. We could demonstrate the viral persistence of BTV-25-GER2018 in goats for up to 4.5 years, and the first BTV-25 isolate is now available for further characterization.

BlueTYPE - A low density TaqMan-RT-qPCR array for the identification of all 24 classical Bluetongue virus serotypes.

Bluetongue virus is a double-stranded RNA virus with 10 genome segments. VP2 is the primary target for neutralising antibodies and defines the serotype. Today, more than 27 serotypes are known, 24 are defined as "classical", and new serotypes are under investigation. Beside group-specific BTV-genome detection, additional serotype characterisation is important for disease control and epidemiological investigations. Therefore, a low-density RT-qPCR array representing a panel of group- and serotype-specific assays, was combined with an internal control system. For BTV serotype detection, both published and the newly developed in-house PCR systems were combined. The different primer-probe-mixes were placed in advance into a 96-well plate stored at -20 °C until use. At the time of analysis, the only template RNA was added to the prepared primer-probe-mixes and heat denatured at 95 °C for 3 min. After cooling, the master mix was added to each well and the PCR could run for around 90 min. The presented low-density TaqMan-RT-qPCR array enables fast and precise characterisation of the BTV serotype in clinical cases. Furthermore, mixed infections can be easily identified. In addition, the newly developed low-density RT-qPCR-array can easily be adapted to novel BTV strain variants or extended for relevant differential diagnosis.

Putative Novel Serotypes '33' and '35' in Clinically Healthy Small Ruminants in Mongolia Expand the Group of Atypical BTV.

Between 2015 and 2018, we identified the presence of three so-far-unknown Bluetongue virus (BTV) strains (BTV-MNG1/2018, BTV-MNG2/2016, and BTV-MNG3/2016) circulating in clinical healthy sheep and goats in Mongolia. Virus isolation from EDTA blood samples of BTV-MNG1/2018 and BTV-MNG3/2016 was successful on the mammalian cell line BSR using blood collected from surveillance. After experimental inoculation of goats with BTV-MNG2/2016 positive blood as inoculum, we observed viraemia in one goat and with the EDTA blood of the experimental inoculation, the propagation of BTV-MNG2/2016 in cell culture was successful on mammalian cell line BSR as well. However, virus isolation experiments for BTV-MNG2/2016 on KC cells were unsuccessful. Furthermore, we generated the complete coding sequence of all three novel Mongolian strains. For atypical BTV, serotyping via the traditional serum neutralization assay is not trivial. We therefore sorted the 'putative novel atypical serotypes' according to their segment-2 sequence identities and their time point of sampling. Hence, the BTV-MNG1/2018 isolate forms the 'putative novel atypical serotype' 33, the BTV-MNG3/2016 the 'putative novel atypical serotype' 35, whereas the BTV-MNG2/2016 strain belongs to the same putative novel atypical serotype '30' as BTV-XJ1407 from China.

BTV antibody longevity in cattle five to eight years post BTV-8 vaccination.

The Bluetongue virus serotype -8 (BTV-8) epizootic in Germany (2006-2008) was successfully eradicated, essentially by the massive application of commercially available inactivated BTV-8 vaccines. While a six-year antibody longevity of BTV antibodies post BTV-8 vaccination in cattle has been described previously, our study investigated the BTV-8-vaccine antibodies in cattle for up to eight years. In total, 157 bovine serum samples were analysed for the presence of group-specific BTV antibodies in both a commercial cELISA, and a BTV-8- specific serum neutralization test. A robust number of cattle were seropositive for group- and serotype-specific neutralising antibodies for five or more years. In selected animals, born and vaccinated in 2009 or later, the presence of BTV antibodies for up to eight years post BTV-8 vaccination could be confirmed. Our data also show, that booster vaccination prolonged the antibody longevity of vaccine-induced antibodies and the number of serologically positive cattle.

Facile synthesis of chrysin-derivatives with promising activities as aromatase inhibitors.

Flavones such as chrysin show structural similarities to androgens, the substrates of human aromatase, which converts androgens to estrogens. Aromatase is a key target in the treatment of hormone-dependent tumors, including breast cancer. Flavone-based aromatase inhibitors are of growing interest, and chrysin in particular provides a (natural) lead structure. This paper reports multicomponent synthesis as a means for facile modification of the chrysin core structure in order to add functional elements. A Mannich-type reaction was used to synthesize a range of mono- and disubstituted chrysin derivatives, some of which are more effective aromatase inhibitors than the benchmark compound, aminoglutethimide. Similarly, the reaction of chrysin with various isonitriles and acetylene dicarboxylates results in a new class of flavone derivatives, tricyclic pyrano-flavones which also inhibit human aromatase. Multicomponent reactions involving flavones therefore enable the synthesis of a variety of derivatives, some of which may be useful as anticancer agents.

Synthesis and antineoplastic activity of O-alkylated derivatives of 7-hydroximinoandrost-5-ene steroids.

Varied positioning of the hydroximino group on the parental steroid skeleton results in remarkable changes in the antineoplastic activity profile of the compounds. Here, the compound 7-oximino-5-androstene and its O-alkylated derivatives have been prepared and screened for cytotoxic and aromatase inhibitory activity. The steroidal 7-oximino ether derivatives exhibited insignificant cytotoxic effects when screened against three cancer cell lines, MCF-7 (breast), NCl-H460 (lung), and SF-268 (CNS) at 100 microM. However, the imidazolyl-substituted steroidal oxime ethers displayed moderate inhibition of cytochrome P450 aromatase.

Selective aldosterone synthase inhibitors reduce aldosterone formation in vitro and in vivo.

Aldosterone plays a crucial role in salt and water homeostasis but in case of pathologically increased plasma aldosterone levels it is also involved in the development and the progression of severe cardiovascular diseases like heart failure and myocardial fibrosis. For the treatment of these diseases we propose inhibition of the aldosterone forming enzyme CYP11B2 as a new pharmacological strategy. We recently developed in vitro highly potent and selective inhibitors of human CYP11B2, but the evidence of their in vivo activity is still missing. For this purpose, rat aldosterone synthase gene was cloned and expressed in V79MZ cells to establish a new screening assay for the identification of "rat-active" substances. Compound 7 from the class of heteroaryl substituted 3,4-dihydro-1H-quinolin-2-ones showed a moderate inhibitory effect (65% at 2 microM) on rat CYP11B2 in vitro. Furthermore, it diminished the conversion of deoxycorticosterone to aldosterone in rat adrenals and significantly reduced plasma aldosterone levels in vivo.

In vivo active aldosterone synthase inhibitors with improved selectivity: lead optimization providing a series of pyridine substituted 3,4-dihydro-1H-quinolin-2-one derivatives.

Pyridine substituted naphthalenes (e.g., I-III) constitute a class of potent inhibitors of aldosterone synthase (CYP11B2). To overcome the unwanted inhibition of the hepatic enzyme CYP1A2, we aimed at reducing the number of aromatic carbons of these molecules because aromaticity has previously been identified to correlate positively with CYP1A2 inhibition. As hypothesized, inhibitors with a tetrahydronaphthalene type molecular scaffold (1-11) exhibit a decreased CYP1A2 inhibition. However, tetralone 9 turned out to be cytotoxic to the human cell line U-937 at higher concentrations. Consequent structural optimization culminated in the discovery of heteroaryl substituted 3,4-dihydro-1H-quinolin-2-ones (12-26), with 12, a bioisostere of 9, being nontoxic up to 200 microM. The investigated molecules are highly selective toward both CYP1A2 and a wide range of other cytochrome P450 enzymes and show a good pharmacokinetic profile in vivo (e.g., 12 with a peroral bioavailability of 71%). Furthermore, isoquinoline derivative 21 proved to significantly reduce plasma aldosterone levels of ACTH stimulated rats.

Novel aldosterone synthase inhibitors with extended carbocyclic skeleton by a combined ligand-based and structure-based drug design approach.

Pharmacophore modeling of a series of aldosterone synthase (CYP11B2) inhibitors triggered the design of compounds 11 and 12 by extending a previously established naphthalene molecular scaffold (e.g., present in molecules 1 and 2) via introduction of a phenyl or benzyl residue in 3-position. These additional aromatic moieties have been hypothesized to fit into the newly identified hydrophobic pharmacophore feature HY3. Subsequent docking studies in our refined CYP11B2 protein model have been performed prior to synthesis to estimate the inhibitory properties of the proposed molecules. While phenyl-substituted compound 11 (IC50 > 500 nM) did not dock under the given pharmacophore constraint (i.e., the Fe(heme)-N(ligand) interaction), benzyl-substituted compound 12 (IC50 = 154 nM) was found to exploit a previously unexplored subpocket of the inhibitor binding site. By structural optimization based on the pharmacophore hypothesis, 25 novel compounds were synthesized, among them highly potent CYP11B2 inhibitors (e.g., 17, IC50 = 2.7 nM) with pronounced selectivity toward the most important steroidogenic and hepatic CYP enzymes.

Overcoming undesirable CYP1A2 inhibition of pyridylnaphthalene-type aldosterone synthase inhibitors: influence of heteroaryl derivatization on potency and selectivity.

Recently, we reported on the development of potent and selective inhibitors of aldosterone synthase (CYP11B2) for the treatment of congestive heart failure and myocardial fibrosis. A major drawback of these nonsteroidal compounds was a strong inhibition of the hepatic drug-metabolizing enzyme CYP1A2. In the present study, we examined the influence of substituents in the heterocycle of lead structures with a naphthalene molecular scaffold to overcome this unwanted side effect. With respect to CYP11B2 inhibition, some substituents induced a dramatic increase in inhibitory potency. The methoxyalkyl derivatives 22 and 26 are the most potent CYP11B2 inhibitors up to now (IC50 = 0.2 nM). Most compounds also clearly discriminated between CYP11B2 and CYP11B1, and the CYP1A2 potency significantly decreased in some cases (e.g., isoquinoline derivative 30 displayed only 6% CYP1A2 inhibition at 2 microM concentration). Furthermore, isoquinoline derivative 28 proved to be capable of passing the gastrointestinal tract and reached the general circulation after peroral administration to male Wistar rats.