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Dengue fever is a severe, widespread, and neglected disease with more than 2 million diagnosed infections per year. The dengue virus NS2B/NS3 protease (PR) represents a prime target for rational drug design. At the moment, there are no clinical PR inhibitors (PIs) available. We have identified diaryl (thio)ethers as candidates for a novel class of PIs. Here, we report the selective and noncompetitive inhibition of the serotype 2 and 3 dengue virus PR in vitro and in cells by benzothiazole derivatives exhibiting 50% inhibitory concentrations (IC 50 s) in the low-micromolar range. Inhibition of replication of DENV serotypes 1 to 3 was specific, since all substances influenced neither hepatitis C virus (HCV) nor HIV-1 replication. Molecular docking suggests binding at a specific allosteric binding site. In addition to the in vitro assays, a cell-based PR assay was developed to test these substances in a replication-independent way. The new compounds inhibited the DENV PR with IC 50 s in the low-micromolar or submicromolar range in cells. Furthermore, these novel PIs inhibit viral replication at submicromolar concentrations. Dengue viruses (DENVs) are enveloped positive-strand RNA viruses and belong to the family Flaviviridae. DENV is the most important arthropod-borne viral infection. Over one-third of the world population lives in areas of DENV endemicity, and an estimated 390 million infections occur every year. In addition, the number of countries having experienced DENV epidemics has risen from 9 in 1970 to more than 100 today (1, 2). Furthermore, the number of diagnosed infections across America, Southeast Asia, and the Western Pacific nearly doubled from 1.2 million in 2008 to over 2.3 million in 2010 (2). Four different DENV serotypes have been identified so far. Recently, evidence for an additional subtype has been presented (3). Serotypes 1 to 4 are now prevalent in Asia, Africa, and America, and the regions where dengue is endemic are still increasing (4-6), with dengue endangering even Europe and the United States due to vector spread. DENV infections can be associated with dengue fever, but up to 88% of the infections remain inapparent (7). These nonpersistent infected patients serve besides persistently infected mosquitoes as a virus reservoir. Severe DENV infections and especially reinfections may lead to dengue hemorrhagic fever and dengue shock syndrome, with lethality up to 5% (2,8,9). There is neither a vaccination nor a specific treatment for DENV infections.The DENV genome contains a single open reading frame, which encodes the structural proteins capsid, membrane precursor (prM), and envelope and the nonstructural proteins NS1, NS2, NS3, NS4, and NS5 (10). Cellular proteases and the viral serine protease (PR) are responsible for cleaving the viral precursor polyprotein into functional proteins. The DENV PR consists of the amino-terminal domain of the NS3 protein and requires NS2B, a 14-kDa protein, as a cofactor to form a stable complex. This heterodimeric PR cleaves at the capsid-prM, NS2A/NS2...

Between 1999 and 2006, 15 cats were diagnosed with disease attributable to a novel mycobacterial species. The infections consisted of granulomatous lesions in the skin, subcutis, and ocular or periocular tissues with an indolent but progressive clinical course. Lesions typically were found in facial regions or on the distal limbs. Cats of all ages and both sexes were affected. Infections often were challenging to treat, although they could be cured using surgery in concert with combination antimicrobial therapy. Microscopically, lesions were granulomatous to pyogranulomatous and contained numerous acid-fast bacilli. Scanty cultures of the causal microorganisms occasionally could be obtained in mycobacterial broth, but subculture to solid media failed. When cultures were not available, DNA was extracted from fresh tissue, lyophilized material, and formalinfixed, paraffin-embedded tissues from lesions. PCR amplification of the 5 end of the 16S rRNA gene and regions within four additional loci (ITS1, hsp65, rpoB, and sodA) was performed with various efficiencies using mycobacterial primers. Nucleotide sequences were unique for each locus tested. Nucleotide sequences obtained from individual cases were identical for each locus for which the amplification was successful. Phylogenetic analysis performed using concatenated partial 16S rRNA and hsp65 gene sequences indicated that this novel mycobacterial species from Victoria is a member of the Mycobacterium simiae-related group, taxonomically related to the mycobacterium causing leproid granulomas in dogs throughout the world. Based on the clustering of cases, we refer to this novel species as Mycobacterium sp. strain Tarwin.

The development and validation of a comprehensive panel of PCR diagnostic tests, predominantly for viruses causing equine respiratory disease, that can be completed within 8 hours from receipt of clinical samples, provides a major advance in the rapid diagnosis or exclusion diagnosis of these endemic equine virus diseases in Australia.

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