No abstract

Original genome annotations need to be regularly updated if the information they contain is to remain accurate and relevant. Here the complete reannotation of the genome sequence of Mycobacterium tuberculosis strain H37Rv is presented almost 4 years after the first submission. Eighty-two new protein-coding sequences (CDS) have been included and 22 of these have a predicted function. The majority were identified by manual or automated reanalysis of the genome and most of them were shorter than the 100 codon cutoff used in the initial genome analysis. The functional classification of 643 CDS has been changed based principally on recent sequence comparisons and new experimental data from the literature. More than 300 gene names and over 1000 targeted citations have been added and the lengths of 60 genes have been modified. Presently, it is possible to assign a function to 2058 proteins (52 % of the 3995 proteins predicted) and only 376 putative proteins share no homology with known proteins and thus could be unique to M. tuberculosis.

Mycobacterium ulcerans (MU), an emerging human pathogen harbored by aquatic insects, is the causative agent of Buruli ulcer, a devastating skin disease rife throughout Central and West Africa. Mycolactone, an unusual macrolide with cytotoxic and immunosuppressive properties, is responsible for the massive s.c. tissue destruction seen in Buruli ulcer. Here, we show that MU contains a 174-kb plasmid, pMUM001, bearing a cluster of genes encoding giant polyketide synthases (PKSs), and polyketide-modifying enzymes, and demonstrate that these are necessary and sufficient for mycolactone synthesis. This is a previously uncharacterized example of plasmid-mediated virulence in a Mycobacterium, and the emergence of MU as a pathogen most likely reflects the acquisition of pMUM001 by horizontal transfer. The 12-membered core of mycolactone is produced by two giant, modular PKSs, MLSA1 (1.8 MDa) and MLSA2 (0.26 MDa), whereas its side chain is synthesized by MLSB (1.2 MDa), a third modular PKS highly related to MLSA1. There is an extreme level of sequence identity within the different domains of the MLS cluster (>97% amino acid identity), so much so that the 16 ketosynthase domains seem functionally identical. This is a finding of significant consequence for our understanding of polyketide biochemistry. Such detailed knowledge of mycolactone will further the investigation of its mode of action and the development of urgently needed therapeutic strategies to combat Buruli ulcer.A single Buruli ulcer, which can cover Ͼ15% of a person's skin surface, contains huge numbers of extracellular bacteria. Despite their abundance and extensive tissue damage, there is a remarkable absence of an acute inflammatory response to the bacteria, and the lesions are often painless (1). This unique pathology is attributed to mycolactone, a macrolide toxin consisting of a polyketide side chain attached to a 12-membered core that seems to have cytotoxic, analgesic, and immunosuppressive activities. Its mode of action is unclear, but, in a guinea pig model of the disease, purified mycolactone injected s.c. reproduces the natural pathology, and mycolactone negative variants are avirulent, implying a key role for the toxin in pathogenesis (2).Mycobacterium ulcerans (MU) and Mycobacterium marinum (MM) share over 98% DNA sequence identity, they occupy aquatic environments, and both cause cutaneous infections (3). However, MM produces a granulomatous intracellular lesion, typical for pathogenic mycobacteria and totally distinct from Buruli ulcer in which MU are mainly found extracellularly. The fact that MM does not produce mycolactone suggested that it might be possible to identify genes for mycolactone synthesis by performing genomic subtraction experiments between MU and MM. Fragments of MU-specific polyketide synthase (PKS) genes were identified from these experiments (4). The subsequent investigation of these sequences led to the discovery of the MU virulence plasmid pMUM001 and the extraordinary PKS locus it encodes. Plasmid Sequence Determination....

†These authors contributed equally to this publication.Dengue virus nonstructural protein 5 (NS5) is a large multifunctional protein with a central role in viral replication. We previously identified two nuclear localization sequences (NLSs) within the central region of dengue virus type-2 (DENV-2) NS5 ('aNLS' and 'bNLS') that are recognized by the importin a/b and importin b1 nuclear transporters, respectively. Here, we demonstrate the importance of the kinetics of NS5 nuclear localization to virus production for the first time and show that the aNLS is responsible. Site-specific mutations in the bipartitetype aNLS or bNLS region were introduced into a reporter plasmid encoding green fluorescent protein fused to the N-terminus of DENV-2 NS5, as well as into DENV-2 genomic length complementary DNA. Mutation of basic residues in the highly conserved region of the bNLS did not affect nuclear import of NS5. In contrast, mutations in either basic cluster of the aNLS decreased NS5 nuclear accumulation and reduced virus production, with the greatest reduction observed for mutation of the second cluster (K 387 K 388 K 389 ); mutagenesis of both clusters abolished NS5 nuclear import and DENV-2 virus production completely. The latter appeared to relate to the impaired ability of virus lacking nuclear-localizing NS5, as compared with wild-type virus expressing nuclear-localizing NS5, to reduce interleukin-8 production as part of the antiviral response. The results overall indicate that NS5 nuclear localization through the aNLS is integral to viral infection, with significant implications for other flaviviruses of medical importance, such as yellow fever and West Nile viruses.

The protein NS3 of Dengue virus type 2 (DEN-2) is the second largest nonstructural protein specified by the virus and is known to possess multiple enzymatic activities, including a serine proteinase located in the Nterminal region and an NTPase-helicase in the remaining 70% of the protein. The latter region has seven conserved helicase motifs found in all members of the family Flaviviridae. DEN-2 NS3 lacking the proteinase region was synthesized as a fusion protein with glutathione S-transferase in Escherichia coli. The effects of 10 mutations on ATPase and RNA helicase activity were examined. Residues at four sites within enzyme motifs I, II, and VI were substituted, and six sites outside motifs were altered by clustered charged-to-alanine mutagenesis. The mutations were also tested for their effects on virus replication by incorporation into genomic-length cDNA. Two mutations, both in motif I (G198A and K199A) abolished both ATPase and helicase activity. Two further mutations, one in motif VI (R457A,R458A) and the other a clustered charged-to-alanine substitution at R 376 KNGK 380 , abolished helicase activity only. No virus was detected for any mutation which prevented helicase activity, demonstrating the requirement of this enzyme for virus replication. The remaining six mutations resulted in various levels of enzyme activities, and four permitted virus replication. For the two nonreplicating viruses encoding clustered changes at R 184 KR 186 and D 436 GEE 439 , we propose that the substituted residues are surface located and that the viruses are defective through altered interaction of NS3 with other components of the viral replication complex. Two of the replicating viruses displayed a temperature-sensitive phenotype. One contained a clustered mutation at D 334 EE 336 and grew too poorly for further characterization. However, virus with an M283F substitution in motif II was examined in a temperature shift experiment (33 to 37°C) and showed reduced RNA synthesis at the higher temperature.The four serotypes of Dengue virus (types 1 to 4) belong to the family Flaviviridae, which consists of the genera Flavivirus, Pestivirus, and Hepacivirus (52). The dengue virus genome is positive-sense RNA of 11 kb and encodes the proteins C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5 in a single open reading frame. Co-and posttranslational polyprotein processing by host and viral proteinases generates three structural proteins, namely, C (capsid), M (membrane associated) and E (envelope), and seven nonstructural (NS) proteins, NS1 through NS5 (reviewed in reference 45). Biochemical functions have been demonstrated for some nonstructural proteins. NS5 possesses RNA-dependent RNA polymerase activity (49). A complex of NS2B and NS3 acts as a chymotrypsin-like serine proteinase; the N-terminal 30% of NS3 is sufficient for this activity (15, 42). The C-terminal 70% of NS3 has seven motifs characteristic of RNA helicases of the DExH subfamily. Recombinant proteins containing the C-terminal helicase region of dengue virus NS3 possess n...

A genomic-length cDNA clone corresponding to the RNA of dengue virus type 2 (DEN-2) New Guinea C strain (NGC) was constructed in a low copy number vector. The cloned cDNA was stably propagated in Escherichia coli and designated pDVWS501. RNA transcripts produced in vitro from the cDNA using T7 RNA polymerase yielded infectious virus (MON501) upon electroporation into BHK-21 cells. When compared with parental NGC virus, MON501 replicated to similar levels in Aedes albopictus C6/36 cells and showed similar neurovirulence in suckling mice. In contrast, a second genomic-length cDNA clone (pDVWS310) used as an intermediate in the construction of pDVWS501 produced virus

Influenza vaccination using the NP appeared to be safe, and acceptable in this first time in humans study, and induced similar immune responses to vaccination by IM injection.

Background The Vaxxas high-density microarray patch (HD-MAP) consists of a high density of microprojections coated with vaccine for delivery into the skin. Microarray patches (MAPs) offer the possibility of improved vaccine thermostability as well as the potential to be safer, more acceptable, easier to use, and more cost-effective for the administration of vaccines than injection by needle and syringe (N&S). Here, we report a phase I trial using the Vaxxas HD-MAP to deliver a monovalent influenza vaccine that was to the best of our knowledge the first clinical trial to evaluate the safety, tolerability, and immunogenicity of lower doses of influenza vaccine delivered by MAPs. Methods and findings HD-MAPs were coated with a monovalent, split inactivated influenza virus vaccine containing A/Singapore/GP1908/2015 H1N1 haemagglutinin (HA). Between February 2018 and March 2018, 60 healthy adults (age 18-35 years) in Melbourne, Australia were enrolled into part A of the study and vaccinated with either: HD-MAPs delivering 15 μg of A/Singapore/ GP1908/2015 H1N1 HA antigen (A-Sing) to the volar forearm (FA); uncoated HD-MAPs; intramuscular (IM) injection of commercially available quadrivalent influenza vaccine (QIV) containing A/Singapore/GP1908/2015 H1N1 HA (15 μg/dose); or IM injection of H1N1 HA antigen (15 μg/dose). After 22 days' follow-up and assessment of the safety data, a further 150 healthy adults were enrolled and randomly assigned to 1 of 9 treatment groups.