Ribavirin antagonizes the effect of azidothymidine on HIV replication.

Azidothymidine and ribavirin both inhibit replication of human immunodeficiency virus in vitro and show promise of clinical utility in patients infected with this virus. In this study, the possible interactions of these drugs were examined in vitro, and a reproducible antagonism between azidothymidine and ribavirin was found to occur under a variety of experimental conditions. The mechanism responsible for this antagonism appeared to be inhibition of azidothymidine phosphorylation by ribavirin. Because similar effects may occur in vivo, clinical trials of these two drugs in combination must be performed only under carefully controlled conditions.

Molecular characterization of a novel fastidious mycobacterium causing lepromatous lesions of the skin, subcutis, cornea, and conjunctiva of cats living in Victoria, Australia.

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 formalin-fixed, 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.

Autochthonous feline leprosy caused by Mycobacterium sp. strain Tarwin affecting a cat from the Central Coast of New South Wales.

CASE REPORT: A 5-year-old Domestic Shorthair-cross was presented with a raised, alopecic skin nodule affecting the external surface of the right upper lip with an adjacent second smaller satellite lesion. Fine needle aspiration cytology revealed numerous intracellular and extracellular negatively stained bacilli. Histopathology confirmed granulomatous inflammation with multinucleate giant cell formation and abundant intracellular acid-fast bacilli, consistent with a mycobacterial aetiology. PCR testing of the fresh tissue from the satellite lesion and subsequent sequence analysis identified Mycobacterium sp. strain Tarwin. The skin lesion was surgically excised and clarithromycin 62.5 mg twice daily was administered to the cat for 25 days. CONCLUSION: There was no recurrence of the lesion at the time of writing, 16 months after the surgery. This is the second autochthonous case of feline leprosy caused by M. sp. strain Tarwin originating in New South Wales, Australia.

Nucleotide sequence and expression in Escherichia coli of the recA gene of Neisseria gonorrhoeae.

The nucleotide sequence of the recA gene of Neisseria gonorrhoeae MS11 has been determined. The product of this gene can act as a recombinase in Escherichia coli, but does so with a decreased efficiency, probably because of the formation of mixed multimers with the equivalent E. coli protein.

The normally silent sigma54 promoters upstream of the pilE genes of both Neisseria gonorrhoeae and Neisseria meningitidis are functional when transferred to Pseudomonas aeruginosa.

The pilE gene encodes the pilin subunit in Neisseria gonorrhoeae and Neisseria meningitidis. Transcriptional analysis of promoters upstream of pilE in N. gonorrhoeae has been described previously (Fyfe et al. (1995) J. Bacteriol. 177, 3781-3787). Transcription from the sigma54-dependent promoter P3 was detected in Pseudomonas aeruginosa. Here we show that this transcription is dependent on the P. aeruginosa transcriptional activator PilR, and a specific upstream sequence with a high degree of similarity to the PilR-binding site found upstream of the P. aeruginosa pilin gene. This implies there is an upstream activator site (UAS) present 5' of pilE. Sequencing upstream of the N. meningitidis MC58 c2 pilE gene shows this region to be very similar to that in N. gonorrhoeae. P3 and the UAS are conserved, although insertions were noted on either side of the UAS. Transcriptional analysis has shown that the N. meningitidis P3 promoter is used in P. aeruginosa, provided PilR and an upstream region that includes sequence similar to the UAS are present. Transcription from the N. meningitidis PpilE is stronger than from the N. gonorrhoeae equivalent. N. meningitidis uses the sigma70 promoter P1 to transcribe pilE.

Neisseria gonorrhoeae contains multiple copies of a gene that may encode a site-specific recombinase and is associated with DNA rearrangements.

A 960-bp ORF potentially encoding a site-specific recombinase has been cloned from Neisseria gonorrhoeae MS11-A. This ORF was designated pivNg on the basis of similarity of the deduced amino acid sequence to the Piv proteins of Moraxella spp. that are site-specific invertases. Southern hybridization and sequence analysis revealed that there were multiple copies of pivNg sequence within the genomes of N. gonorrhoeae strains tested, but not in several other neisserial species. Southern hybridization and sequence analysis further suggested that pivNg sequences may be associated with genomic rearrangements.

Control of gonococcal pilin-encoding gene expression in Escherichia coli.

The pilE gene from Neisseria gonorrhoeae, unlike other type-4 pilin-encoding genes, is well expressed in Escherichia coli. Two putative promoters have been implicated in the transcription of this gene. Besides the -24/-12 promoter used to transcribe type-4 pilin-encoding genes in most species, the consensus sequence for a conventional promoter is also present. The two promoters overlap and would have almost identical transcription start points (tsp). Transcription from a -24/-12 promoter should be abolished in an E. coli rpoN mutant. A recombinant plasmid carrying pilE could not be transformed into such a mutant, apparently because the synthesis of the N-terminal hydrophobic domain of pilin is lethal to the rpoN mutant. This suggests that pilE is expressed at a higher level in an rpoN mutant than it is in a wild-type (wt) strain of E. coli. This suggestion was confirmed by constructing fusions between the pilE promoter region and a promoter-less cat gene. We suggest that the conventional promoter is primarily responsible for the transcription of pilE, but that the binding of the RpoN sigma factor partially represses transcription of this gene in wt strains. In an rpoN mutant, the repression is removed and transcription occurs at a level that is lethal to the mutant host.

Absence of an SOS-like system in Neisseria gonorrhoeae.

The DNA repair capacities of Neisseria gonorrhoeae have not been well characterised, however, it is known that the gonococcus possesses an excision repair system. The fact that genes in this system are part of the SOS regulon in Escherichia coli prompted this investigation into the transcriptional regulation of genes involved in DNA repair in N. gonorrhoeae. Northern (RNA-DNA) dot blot hybridisation was used to investigate potential DNA damage-mediated induction of the gonococcal recA, uvrA and uvrB genes. In contrast to the situation in E. coli, transcription of these genes in N. gonorrhoeae was not induced in response to treatment with methyl methanesulfonate (MMS) and UV light. These data indicated that the gonococcus does not possess an SOS-like system that is induced in response to DNA damage.

Transcriptional analysis of the groESL operon of Neisseria gonorrhoeae.

The nucleotide sequence upstream of the groEL gene of Neisseria gonorrhoeae has been determined. Upstream of groEL is a homolog of groES and the divergently transcribed frpB gene. The promoter region of groES lacks the inverted repeat sequences (IR) that act as a regulatory element controlling the expression of similar operons in many other bacterial species. This region contains overlapping consensus sequences for sigma 32-dependent and sigma 70-dependent promoters, and an appropriately placed transcription start point was mapped downstream of these promoters. Northern hybridization demonstrated that synthesis of a full-length groES-groEL transcript was induced in heat-stressed cells. These experiments also revealed the presence of a shorter groES-specific transcript, apparently the result of the premature termination of transcription at an IR situated between the groES and groEL genes.

The pathogenic neisseriae contain an inactive rpoN gene and do not utilize the pilE sigma54 promoter.

The sigma54 promoter (P3) upstream of the pilE gene in Neisseria gonorrhoeae was shown to be non-functional by transcriptional analysis of a PpilE::lacZ fusion containing only P3. A region on the chromosome of N. gonorrhoeae strain MS11-A was identified that potentially encodes a protein with a significant similarity to the Escherichia coli RpoN protein. However, this region (designated RLS for rpoN-like sequence) does not contain a single open reading frame (ORF) capable of encoding a functional RpoN protein. It appears that RLS may have arisen from an ancestral rpoN homologue that underwent a deletion removing the sequence encoding the essential helix-turn-helix (HTH) motif, and changing the subsequent reading frame. An RLS has been identified in several strains of N. gonorrhoeae and N. meningitidis. A 90-kDa gonococcal protein has previously been shown to react with a monoclonal antibody raised against the RpoN from Salmonella typhimurium. However, mutagenesis and Western blot analysis confirmed that the gene encoding this protein is not contained within RLS.

Selection and preliminary characterization of acyclovir-resistant mutants of varicella zoster virus.

A series of acyclovir-resistant mutants of varicella zoster virus (VZV) were selected in vitro by serial passage of VZV-infected human fibroblasts in increasing drug concentrations, or by continuous exposure of cultures infected at high multiplicity to 100 microM acyclovir. The in vitro susceptibility of these mutants to several antiherpetic agents was measured by the plaque-reduction assay. The capacity of extracts of cells infected with these mutants to phosphorylate acyclovir was examined and compared with that of their acyclovir-sensitive parent strains. Based on these studies, VZV could be shown to acquire resistance to acyclovir through diminished acyclovir phosphorylation. This was presumable due to loss of viral specific thymidine kinase (TK) function. Two acyclovir-resistant mutants remained TK competent but demonstrated phenotypic changes in sensitivity to antiviral agents known to act at the herpes simplex virus (HSV)-specific DNA polymerase level. These results suggest that the resistance of VZV to acyclovir results from qualitative or quantitative alterations in the virus-specified TK or DNA polymerase.

Activation and antiviral effect of acyclovir in cells infected with a varicella-like simian virus.

Acyclovir inhibited the replication of a varicella-like simian virus (DHV-1) in cell culture (Vero cells) with an ED50 of 38 +/- 2 microM. The activation of acyclovir in this cell culture system was compared with that in the cell system with human varicella zoster virus (VZV). Extracts of cells infected with DHV-1 catalyzed the phosphorylation of acyclovir. The phosphorylation was inhibited by dThd, suggesting the catalyst was a dThd kinase. Electrophoresis of cytosol fractions on polyacrylamide gels corroborated the existence of a virus-associated dThd kinase. This enzyme copurified with an acyclovir-phosphorylating activity. The enzyme catalyzed the phosphorylation of acyclovir at a greater relative rate than that with the VZV enzyme, but with a higher apparent Km value for acyclovir. The relative efficiencies for the two enzymes with acyclovir were similar. Anabolic studies with cells infected with DHV-1 and incubated with [14C]acyclovir indicated that triphosphate of acyclovir did accumulate. The results indicate that acyclovir is activated in cells infected with DHV-1 in a manner similar to that in cells infected with VZV.

Modifications on the heterocyclic base of acyclovir: syntheses and antiviral properties.

A group of compounds was prepared in which variations of the ring portion of the acyclovir (ACV) structure were made. These modifications included monocyclic (isocytosine, triazole, imidazole), bicyclic (8-azapurine, pyrrolo[2,3-d]pyrimidine, pyrazolo[3,4-d]pyrimidine) and tricyclic (linear benzoguanine) congeners. The derivatives were evaluated against herpes simplex virus type 1 (HSV-1) by the plaque-inhibition and plaque-reduction methods with only the 8-azapurine analogue 28 showing some activity. In a test measuring the ability of these compounds to inhibit the HSV-1 thymidine kinase, 28 and the tricyclic derivative 38 exhibited competition with ACV for binding to the enzyme. The inability of the group to exert significant antiherpetic action is attributed to their lack of phosphorylation to the requisite triphosphate stage.

3-Deazaadenosine 5'-triphosphate: a novel metabolite of 3-deazaadenosine in mouse leukocytes.

Evidence has been obtained for the metabolic formation of small amounts (1-2% of the ATP pool) of 3-deazaadenosine 5'-triphosphate (c3ATP) from 3-deazaadenosine (c3Ado) in mouse cytolytic lymphocytes and mouse resident peritoneal macrophages. With intact leukocytes, pharmacological evidence was obtained that adenosine kinase was not the enzyme chiefly responsible for the phosphorylation of c3Ado. Moreover, in the presence of MgCl2, NaCl and IMP, purified rat liver 5'-nucleotidase catalyzed the phosphorylation of c3Ado to 3-deazaadenosine 5'-monophosphate (c3AMP). Two lines of evidence suggest that the metabolic formation of c3ATP is not involved in the inhibition of leukocyte function caused by c3Ado. First, the inhibitory action of c3Ado on antibody-dependent phagocytosis and lymphocyte-mediated cytolysis was reversed markedly upon removal of the drug from the medium. However, the intracellular content of c3ATP remained constant in lymphocytes and macrophages after removal of c3Ado. Second, in macrophages and in lymphocytes, similar intracellular amounts of c3ATP were formed from both c3Ado and 3-deazaadenine under conditions in which the former was biologically active and the latter was essentially inactive. Thus, it appears unlikely that the novel c3ATP metabolite is of relevance for the mechanism of action of c3Ado in mouse leukocytes.

Varicella-zoster virus thymidine kinase. Characterization and substrate specificity.

The varicella-zoster virus (VZV) thymidine kinase (TK) EC 2.7.2.21) catalyzes the phosphorylation of many anti-VZV nucleosides. Purified, bacterially expressed VZV TK was characterized with regard to N-terminal amino acid sequence, pI value, pH optimum, metal ion requirement, phosphate donor and acceptor specificity, and inhibition by dTTP. Initial velocities of thymidine phosphorylation with variable MgATP concentrations fit a two-site model with apparent Km values for MgATP of 0.10 and 900 microM. dTTP was a noncompetitive inhibitor of thymidine phosphorylation but was competitive with MgATP. Phosphate donor and acceptor specificities of the bacterially expressed enzyme were indistinguishable from those of VZV TK purified from infected cells. Detailed studies of the nucleoside specificity with the bacterially expressed enzyme showed that, for a given sugar moiety, thymine nucleosides were the most efficient substrates followed by nucleosides of cytosine, uracil, adenine, and with some exceptions, guanine. For a given pyrimidine or purine (except guanine), 2'-deoxyribonucleosides were the most efficient substrates, followed by arabinosides, ribonucleosides, 2',3'-dideoxyribonucleosides, and the acyclic moiety of acyclovir.

The genome of Neisseria gonorrhoeae retains the remnants of a two-component regulatory system that once controlled piliation.

An intact activator-binding site upstream of the sigma(54) promoter of the pilin-encoding pilE gene of Neisseria gonorrhoeae suggests gonococci produce a protein capable of binding this sequence. We cloned a chimeric gene, rsp, that has sequence similarity to both the pilS and pilR genes of Pseudomonas aeruginosa encoding a two-component regulatory system that controls piliation. This gene is transcribed in N. gonorrhoeae and indirect evidence suggests that Rsp binds to the activator-binding site of the pilE gene. Despite this, mutation of rsp has no effect on piliation in N. gonorrhoeae, suggesting that the remnants of this regulatory system have persisted in the genome, despite the loss of its original function.

Epidemiology and control of tuberculosis in Victoria, a low-burden state in south-eastern Australia, 2005-2010.

SETTING: Victoria, Australia. OBJECTIVE: To describe the epidemiology and control of tuberculosis (TB) in Victoria, 2005-2010. DESIGN: Retrospective review of laboratory-confirmed TB in Victoria, 2005-2010. State TB reference laboratory records were matched with Department of Health notification records to obtain laboratory, demographic, clinical and treatment data. RESULTS: The incidence of TB fell in the Australian-born population but increased overall, reflecting an increase in the proportion of overseas-born cases from 88.9% to 95.8% between 2005 and 2010 (P = 0.03). Patients from India and Viet Nam accounted for over one third of all cases. For overseas-born cases, the median time between arrival and diagnosis was 4 years. Half of all diagnoses were pulmonary disease, of which 45.4% were Ziehl-Neelsen smear-positive. Treatment was most commonly self-administered (76.9%), and very few patients defaulted or failed treatment (1.1%). Only 4.1% of cases were linked to another laboratory-confirmed case. Multidrug-resistant TB remained uncommon (1.7% of cases). CONCLUSIONS: TB in Victoria remains low by global standards and continues to overwhelmingly affect the overseas-born population. Current TB control strategies in Victoria are effective, but strengthened control in high-burden countries will also improve TB control locally.

Localised Mycobacterium ulcerans infection in four dogs.

Localised infection caused by Mycobacterium ulcerans is described in two Kelpies, a Whippet and a Koolie domiciled on the Bellarine Peninsula, Victoria, Australia. The diagnosis was confirmed using real-time polymerase chain reaction (PCR) targeting the M. ulcerans-specific insertion sequence (IS2404) in DNA extracted from swabs of ulcerated lesions in all cases. Where available, molecular typing confirmed that three of the dogs were infected with a strain of M. ulcerans that was indistinguishable from a disease-causing strain in people and other animals in Victoria. One dog was still undergoing treatment at the time of writing, but the remaining three dogs were successfully treated with a combination of surgical debridement and medical therapy in one case, and medical therapy alone in the other two. Investigation of the home environs of three of the dogs using real-time PCR revealed low amounts of M. ulcerans DNA in various environmental samples. Mycobacterium ulcerans infection should be included in the differential diagnoses of any ulcerated skin lesions in dogs that live in or visit endemic areas of Victoria and Queensland.

Mycobacterium ulcerans infections in two horses in south-eastern Australia.

Two horses were diagnosed as having Mycobacterium ulcerans infections. The first was a 21-year-old Quarterhorse-cross mare living in Mallacoota (a coastal town near the border of New South Wales and Victoria, Australia) that presented with lichenification, hair-loss and oedema on a fetlock, which subsequently ulcerated, as well as a non-healing ulcer on the wither. The second horse was a 32 year-old Standardbred gelding from Nicholson, near Bairnsdale, Victoria, that had an ulcerated lesion on its caudal thigh. Histologically, there were characteristic changes seen with M. ulcerans infections in other species, including extensive necrosis without associated granulomatous inflammation. The organisms were seen in Ziehl-Neelsen-stained smears or sections of the lesions from both horses and were isolated in culture from the first horse. A definitive diagnosis was provided by real-time polymerase chain reaction targeting the M. ulcerans-specific insertion sequence, IS2404. Delayed identification of the infectious agent in the first case led to the use of suboptimal antimicrobial therapy, resulting in failure to control the infection and the horse was subsequently euthanased. The second horse was successfully treated following surgical debulking of the centre of the lesion and one session of aggressive cryosurgery. Mycobacterium ulcerans should be considered in the differential diagnosis of unexplained lichenification with oedematous and ulcerated skin lesions in horses living in regions where this organism is endemic.