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Physical and genetic map of the Wiseana nucleopolyhedrovirus genome.

Wiseana nucleopolyhedrovirus (NPV) is the major pathogen of the New Zealand endemic pasture pest, Wiseana spp. To characterize this potential biological control agent, the genome of a virus isolated from Wiseana signata was purified and cloned. The complete genome was cloned as BamHI or HindIII restriction fragments, which were mapped by Southern hybridization and restriction analysis. To verify the physical map, the junctions between all HindIII fragments were confirmed by sequencing. The viral genome was estimated to be 128 kbp. Sequence data generated at the termini of cloned restriction fragments were compared to sequence databases to identify putative gene homologues. Seventeen putative ORFs, which were homologous to other baculoviral sequences, were identified. These putative ORFs were located on the Wiseana NPV physical map and their distribution was compared to genetic maps of NPVs isolated from Autographa californica, Orgyia pseudotsugata and Lymantria dispar. Although the virus from W. signata was significantly different from these other NPVs, a core region of the viral genome was conserved.

Animals↗

The pathogenic potential of endogenous retroviruses: facts and fantasies.

Endogenous retroviruses are descendants of viruses that became cellular genes by integration into their host's genome. They still contribute to pathogenicity as a partner in recombination events, by de novo insertion after mobilization followed by activation of downstream proto-oncogenes, or by gene disruption. Re-expression of viral proteins accompanied by loss of immune tolerance could induce immune disturbances.

Animals↗

Recognition of single-stranded RNA viruses by Toll-like receptor 7.

Viral infection of mammalian host results in the activation of innate immune responses. Toll-like receptors (TLRs) have been shown to mediate the recognition of many types of pathogens, including viruses. The genomes of viruses possess unique characteristics that are not found in mammalian genomes, such as high CpG content and double-stranded RNA. These genomic nucleic acids serve as molecular signatures associated with viral infections. Here we show that TLR7 recognizes the single-stranded RNA viruses, vesicular stomatitis virus and influenza virus. The recognition of these viruses by plasmacytoid dendritic cells and B cells through TLR7 results in their activation of costimulatory molecules and production of cytokines. Moreover, this recognition required intact endocytic pathways. Mice deficient in either the TLR7 or the TLR adaptor protein MyD88 demonstrated reduced responses to in vivo infection with vesicular stomatitis virus. These results demonstrate microbial ligand recognition by TLR7 and provide insights into the pathways used by the innate immune cells in the recognition of viral pathogens.

Adaptor Proteins, Signal Transducing↗

Complete DNA sequence, comparative genomics, and prevalence of an IncHI2 plasmid occurring among extraintestinal pathogenic Escherichia coli isolates.

We have sequenced a large plasmid that occurs among avian pathogenic Escherichia coli isolates. This plasmid, pAPEC-O1-R, is a 241,387-bp IncHI2 plasmid which is cotransmissible via bacterial conjugation with a ColBM virulence plasmid, encodes resistance to eight antimicrobial agents, and appears to occur at low rates among extraintestinal E. coli isolates.

Animals↗

Novel type IV secretion system involved in propagation of genomic islands.

Type IV secretion systems (T4SSs) mediate horizontal gene transfer, thus contributing to genome plasticity, evolution of infectious pathogens, and dissemination of antibiotic resistance and other virulence traits. A gene cluster of the Haemophilus influenzae genomic island ICEHin1056 has been identified as a T4SS involved in the propagation of genomic islands. This T4SS is novel and evolutionarily distant from the previously described systems. Mutation analysis showed that inactivation of key genes of this system resulted in a loss of phenotypic traits provided by a T4SS. Seven of 10 mutants with a mutation in this T4SS did not express the type IV secretion pilus. Correspondingly, disruption of the genes resulted in up to 100,000-fold reductions in conjugation frequencies compared to those of the parent strain. Moreover, the expression of this T4SS was found to be positively regulated by one of its components, the tfc24 gene. We concluded that this gene cluster represents a novel family of T4SSs involved in propagation of genomic islands.

Bacterial Proteins↗

Identification of acquired DNA in Neisseria lactamica.

Anomalous DNA (aDNA) in prokaryotic genomes, identified by its aberrant nucleotide composition, generally represents horizontally acquired DNA. Previous studies showed that frequent DNA transfer occurs between commensal Neisseriae and Neisseria meningitidis. Currently, it is unknown whether aDNA regions are also transferred between these species. The genome of Neisseria lactamica strain 892586 was assessed by a strategy that enables the selective isolation of aDNA, using endonucleases with recognition sites that are overrepresented in aDNA. Of eight regions with aDNA, five displayed similarity to virulence-associated meningococcal sequences. Of three aDNA fragments with limited or no similarity to neisserial sequences, one encodes a novel putative autotransporter/adhesin. The remaining two fragments are adjacent in the N. lactamica genome, and encode a novel putative ATPase/subtilisin-like protease operon. A similar operon is present in the genomes of different respiratory tract pathogens. The identification of aDNA from N. lactamica with similarity to meningococcal aDNA shows that genetic exchange between the Neisseriae is not limited to the neisserial core genome. The discovery of aDNA in N. lactamica similar to a locus in other pathogens substantially expands the neisserial gene pool.

Adenosine Triphosphatases↗

Cloning of herpesviral genomes as bacterial artificial chromosomes.

Herpesviruses, which are important pathogens for both animals and humans, have large and complex genomes with a coding capacity for up to 225 open reading frames (ORFs). Due to the large genome size and the slow replication kinetics in vitro of some herpesviruses, mutagenesis of viral genes in the context of the viral genome by conventional recombination methods in cell culture has been difficult. Given that mutagenesis of viral genes is the basic strategy to investigate function, many of the herpesvirus ORFs could not be defined functionally. Recently, a completely new approach for the construction of herpesvirus mutants has been developed, based on cloning of the virus genome as a bacterial artificial chromosome (BAC) in E. coli. This technique allows the maintenance of viral genomes as a plasmid in E. coli and the reconstitution of viral progeny by transfection of the BAC plasmid into eukaryotic cells. Any genetic modification of the viral genome in E. coli using prokaryotic recombination proteins is possible, thereby allowing the generation of mutant viruses and facilitating the analysis of herpesvirus genomes cloned as infectious BACs. In this review, we describe the principle of cloning a viral genome as a BAC using murine gammaherpesvirus 68 (MHV-68), a mouse model for gammaherpesvirus infections, as an example.

Animals↗

Bacterial genomics as a potential tool for discovering new antimicrobial agents.

The past 30 years have witnessed the emergence of new infectious diseases as well as the re-emergence of those thought to be defeated or under control. It is likely that this threat will continue and that infectious micro-organisms will be found to be responsible for numerous diseases whose etiology had been previously unknown. Compounding this threat is the rapid evolution of drug resistance by micro-organisms that is rendering many existing antimicrobial agents obsolete. Thus, there is an urgent need for the development of new classes of antimicrobial agents and the identification of new drug targets. Over the past decade, advances in high-throughput automated DNA sequencing have delivered a wealth of genetic information in the form of whole genome sequences of microbial pathogens. Coupled with this advancement has been the development of new genetic tools and computational advances capable of selecting genes of particular interest as well as testing for the effects of candidate drugs. While no new drugs have yet been developed, further study into the application and limitations of these new approaches to the identification of novel targets will aid in overcoming the current problem of antimicrobial drug resistance.

Bacteria↗

Do Taq-generated RT-PCR products from RNA viruses accurately reflect viral genetic heterogeneity?

Since the first report of genetically heterogeneous, or quasispecies, populations of RNA viruses, the genetic heterogeneity of the RNA genomes of major viral pathogens has been extensively studied. These studies aim to provide insights into the evolutionary pressures that act upon viruses, in order to define windows where anti-viral therapies will be most effective, to take prognostic values from viral genetic distributions at a given time, and to aid the development of novel therapeutic compounds that may tilt viral replication towards information loss. Many methodologies are employed to analyse genetic distributions of a virus in a given sample, but all involve the generation, and subsequent analysis, of the sequence information contained in a reverse-transcription-polymerase chain reaction (RT-PCR) product. Despite the fact that the aim of these RT-PCRs is to obtain sequence information from viral genomes, their application to this task is approached without adequate consideration of this end-goal. The establishment of an RT-PCR for a specific viral target genome generally proceeds in the same fashion as one would apply to establishing a PCR to determine the presence or absence of a specific target sequence in a given sample. However, it is becoming increasingly apparent that RT-PCR products generated by amplification with the ubiquitous thermostable DNA polymerase Taq, coupled with standard cloning and sequencing methodologies, has the potential to yield inaccurate and misleading data as pertains to the information content of populations of RNA viral genomes. This review discusses varying approaches employed to analyse heterogeneous populations of hepatitis C virus RNA genomes.

Base Pair Mismatch↗

Chlamydia outer membrane protein discovery using genomics.

Outer membrane proteins of microbial pathogens serve essential roles in engaging the host environment and can be important immunotherapeutic targets. Because of the difficulty of growing large quantities of chlamydiae suitable for biochemical fractionation, little was known about their outer membrane protein composition prior to the recent sequencing of the C. trachomatis and C. pneumoniae genomes. Using bioinformatic approaches to characterize chlamydial open reading frames, novel outer membrane proteins were predicted. Several of the predicted outer membrane proteins recently have been shown to be translated and localized to the surface of the chlamydial outer membrane.

Bacterial Outer Membrane Proteins↗

A family of transcripts (K2) of Entamoeba histolytica contains polymorphic repetitive regions with highly conserved elements.

Sera from patients with invasive amebiasis were used to identify a cDNA clone (K2p-1) encoding a commonly recognized, repeat-containing antigen of the pathogenic Entamoeba histolytica HM-1:IMSS. K2p-1 was used to isolate 3 cDNA clones (K2 clones); one K2p-1 related clone from the same pathogenic E. histolytica strain and 2 from the nonpathogenic E. histolytica strain SAW-142. The nucleotide sequence and predicted amino acid sequence revealed a closely related family of transcripts differing mainly in the extent and arrangement of an internal region consisting of tandemly arranged repetitive elements. The repetitive units encoding either 12 or 8 amino acids were found to be highly conserved in all the K2 clones analyzed so far, suggesting that the repeat motifs perform functions common to both pathogenic and nonpathogenic E. histolytica. The genomic organization of the K2 genes was different when compared in pathogenic and nonpathogenic E. histolytica and may therefore be used to discriminate between pathogenic and nonpathogenic E. histolytica strains.

Amino Acid Sequence↗

Altered levels of Salmonella DNA adenine methylase are associated with defects in gene expression, motility, flagellar synthesis, and bile resistance in the pathogenic strain 14028 but not in the laboratory strain LT2.

Comparative genomic analysis has revealed limited strain diversity between Salmonella pathogenic and nonpathogenic isolates. Thus, some of the relative virulence and host-immune response disparities may be credited to differential gene regulation rather than gross differences in genomic content. Here we show that altered levels of Salmonella DNA adenine methylase (Dam) resulted in acute defects in virulence-associated gene expression, motility, flagellin synthesis, and bile resistance in the Salmonella pathogenic strain 14028 but not in avirulent laboratory strain LT2. The defects in motility exhibited by 14028 in response to altered Dam levels was not dependent on the presence of the regulatory protein, RpoS. The transitioning between flagellar types (phase variation) was also differentially regulated in 14028 versus LT2 in response to dam levels, resulting in distinct differences in flagellin expression states. These data suggest that differential gene regulation may contribute to the relative virulence disparities observed between Salmonella serovars that are closely related at the DNA level.

Bacterial Proteins↗

[Viral therapy of cancer].

In spite of the advances achieved in cancer therapeutics, conventional therapies still face problems such as toxicity to normal tissues and low efficacy in advanced phases of the disease. One of the most recent options, albeit experimental, is gene therapy. This strategy shows higher specificity than conventional therapies, but the low efficiency of gene transduction is a hurdle yet to be overcome, and the clinical trials have not shown the expected efficacy. Recently, oncolytic viruses have revived as an alternative in the field of cancer therapeutics. By virtue of the advances in virology and molecular biology techniques, the manipulation of viral genomes to attenuate their pathogenicity and the modification of their life cycle to allow tumor specific viral replication are now possible. At the moment, adenovirus and herpes simplex type 1 mutants are the most commonly employed viruses. Recent clinical trials using these mutant viruses have shown safety and efficacy. Moreover, viral therapy has been combined with radiotherapy or chemotherapy demonstrating additive antineoplastic effects. In this manuscript, we review the biology behind this therapy, the obstacles, and the ongoing clinical trials.

Animals↗

Genomic and bioinformatics analysis of HAdV-4, a human adenovirus causing acute respiratory disease: implications for gene therapy and vaccine vector development.

Human adenovirus serotype 4 (HAdV-4) is a reemerging viral pathogenic agent implicated in epidemic outbreaks of acute respiratory disease (ARD). This report presents a genomic and bioinformatics analysis of the prototype 35,990-nucleotide genome (GenBank accession no. AY594253). Intriguingly, the genome analysis suggests a closer phylogenetic relationship with the chimpanzee adenoviruses (simian adenoviruses) rather than with other human adenoviruses, suggesting a recent origin of HAdV-4, and therefore species E, through a zoonotic event from chimpanzees to humans. Bioinformatics analysis also suggests a pre-zoonotic recombination event, as well, between species B-like and species C-like simian adenoviruses. These observations may have implications for the current interest in using chimpanzee adenoviruses in the development of vectors for human gene therapy and for DNA-based vaccines. Also, the reemergence, surveillance, and treatment of HAdV-4 as an ARD pathogen is an opportunity to demonstrate the use of genome determination as a tool for viral infectious disease characterization and epidemic outbreak surveillance: for example, rapid and accurate low-pass sequencing and analysis of the genome. In particular, this approach allows the rapid identification and development of unique probes for the differentiation of family, species, serotype, and strain (e.g., pathogen genome signatures) for monitoring epidemic outbreaks of ARD.

Adenovirus Infections, Human↗

Genomic and experimental evidence for a potential sexual cycle in the pathogenic thermal dimorphic fungus Penicillium marneffei.

All meiotic genes (except HOP1) and genes encoding putative pheromone processing enzymes, pheromone receptors and pheromone response pathways proteins in Aspergillus fumigatus and Aspergillus nidulans and a putative MAT-1 alpha box mating-type gene were present in the Penicillium marneffei genome. A putative MAT-2 high-mobility group mating-type gene was amplified from a MAT-1 alpha box mating-type gene-negative P. marneffei strain. Among 37 P. marneffei patient strains, MAT-1 alpha box and MAT-2 high-mobility group mating-type genes were present in 23 and 14 isolates, respectively. We speculate that P. marneffei can potentially be a heterothallic fungus that does not switch mating type.

Amino Acid Sequence↗

Molecular epidemiology and diagnosis of Leishmania: what have we learnt from genome structure, dynamics and function?

This paper reviews our exploration of the dynamics of the Leishmania genome and its contribution to epidemiology and diagnosis. We used as a model Peruvian populations of L. (Viannia) braziliensis and L. (V.) peruviana, 2 species very close phylogenetically, but phenotypically very different in biotope and pathology. We initially focused on karyotype analysis. Our data showed that chromosomes were subject to a fast rate of evolution, and were sensitive indicators of genetic drift. Therefore, molecular karyotyping appeared an adequate tool for monitoring (i) emergence of close species, (ii) ecogeographical differentiation at the intraspecific level, and (iii) strain 'fingerprinting'. Chromosome size variation was mostly due to the number of tandemly repeated genes (rDNA, mini-exon, gp63, and cysteine proteinase genes), and could involve the deletion of unique genes (L. (V.) braziliensis-specific gp63 families). Considering the importance of these genes in parasitism, their rearrangement might have functional implications: adaptation to different environments and pleomorphic pathogenicity. Our knowledge of genome structure and dynamics was used to develop new polymerase chain reaction (PCR) techniques. Amplification of gp63 genes followed by cleavage with restriction enzymes and study of restriction fragment length polymorphism (gp63 PCR-RFLP) allowed the discrimination of all species tested, even directly in biopsies with 95% sensitivity (compared with PCR amplification of kinetoplast deoxyribonucleic acid). At the intra-specific level, RFLP was also observed and corresponded to mutations in major immunogen domains of gp63. These seem to be under strong selection pressure, and the technique should facilitate addressing how the host's immune pressure may modulate parasite population structure. Altogether, gp63 PCR-RFLP represents a significant operational improvement over the other techniques for molecular epidemiology and diagnosis: it combines sensitivity, discriminatory power and prognostic value.

Animals↗

Efficient use of a small genome to generate antigenic diversity in tick-borne ehrlichial pathogens.

Ehrlichiae are responsible for important tick-transmitted diseases, including anaplasmosis, the most prevalent tick-borne infection of livestock worldwide, and the emerging human diseases monocytic and granulocytic ehrlichiosis. Antigenic variation of major surface proteins is a key feature of these pathogens that allows persistence in the mammalian host, a requisite for subsequent tick transmission. In Anaplasma marginale pseudogenes for two antigenically variable gene families, msp2 and msp3, appear in concert. These pseudogenes can be recombined into the functional expression site to generate new antigenic variants. Coordinated control of the recombination of these genes would allow these two gene families to act synergistically to evade the host immune response.

Amino Acid Sequence↗

Genomic and biochemical relatedness between Vibrio cholerae serovar O139 and serovar O1 eltor strains.

Vibrio cholerae O139 (Bengal) the new pandemic cholera strain emerging on the Indian subcontinent has revealed considerable homology to Vibrio cholerae O1 EL Tor (strain of the seventh pandemic cholera) in terms of genetic and biochemical properties. Apart from capsule and O139 LPS formation, all strains of V. cholerae O139 were found to be identical to V. cholerae O1 EL Tor strains with respect to genomic restriction fragment length polymorphism, genomic distribution of the pathogenic island, pattern of OMP and multilocus enzymes. However, the analysis of a nonpathogenic V. cholerae O139 isolate from Sri Lanka with a totally different pattern of genetic properties underline that horizontal gene transfer of a piece of DNA encoding biosynthesis of the Vibrio cholerae O139-specific LPS and capsule formation to an O1 El Tor precursor strains must have occurred giving rise to a kind of hybrid V. cholerae O1 El Tor encoding the new serovar-specific O139 antigens.

Genome, Bacterial↗