MicrobiologyBytes

Studies of virus particles and the steps in their life cycle have spearheaded our understanding of biological systems at the molecular level. These studies, however, relied on virus specimens isolated from nature. This dependency changed forever in 2002 when the chemical synthesis of poliovirus, in the absence of any natural template, was published. The work caused a shock wave because it led to excitement as well as revulsion, reflecting the new reality that, for better or worse, all of the more than 2,000 viruses whose genome sequences are deposited by the National Center for Biotechnology Information can be recreated in the laboratory in the absence of natural isolates. So what have we learned?

Synthetic poliovirus and other designer viruses: what have we learned from them? (2011) Annu Rev Microbiol. 65:583-609
Owing to known genome sequences, modern strategies of DNA synthesis have made it possible to recreate in principle all known viruses independent of natural templates. We describe the first synthesis of a virus (poliovirus) in 2002 that was accomplished outside living cells. We comment on the reaction of laypeople and scientists to the work, which shaped the response to de novo syntheses of other viruses. We discuss those viruses that have been synthesized since 2002, among them viruses whose precise genome sequence had to be established by painstakingly stitching together pieces of sequence information, and viruses involved in zoonosis. Synthesizing viral genomes provides a powerful tool for studying gene function and the pathogenic potential of these organisms. It also allows modification of viral genomes to an extent hitherto unthinkable. Recoding of poliovirus and influenza virus to develop new vaccine candidates and refactoring the phage T7 DNA genome are discussed as examples.

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This review gives an overview of the current state-of-the-art of bacteriophage therapy and discusses the challenges to be overcome before phage therapy can become a part of Western medicine.

The next generation of bacteriophage therapy Curr Opin Microbiol. (2011)14(5): 524-531
Bacteriophage therapy for bacterial infections is a concept with an extensive but controversial history. There has been a recent resurgence of interest into bacteriophages owing to the increasing incidence of antibiotic resistance and virulent bacterial pathogens. Despite these efforts, bacteriophage therapy remains an underutilized option in Western medicine due to challenges such as regulation, limited host range, bacterial resistance to phages, manufacturing, side effects of bacterial lysis, and delivery. Recent advances in biotechnology, bacterial diagnostics, macromolecule delivery, and synthetic biology may help to overcome these technical hurdles. These research efforts must be coupled with practical and rigorous approaches at academic, commercial, and regulatory levels in order to successfully advance bacteriophage therapy into clinical settings.

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The advent of genetic engineering – the ability to edit and insert DNA into living organisms – in the latter half of the 20th century created visions of a new era of synthetic biology, where novel biological functions could be designed and implemented for useful purposes. We are witnessing an exciting revolution of scale, wherein technical progresses allow for the manipulation of genetic material at the whole genome level. This will enable the manufacture of increasingly complex genetic designs to solve pressing challenges in health, energy and the environment-if and when such designs can be specified.

This paper argues that the organized development of key common application organisms, engineered for engineerability, and attendant libraries of parts, pathways and standardized manufacturing are necessary for this genome-scale technology to realize its promise.

Toward rational design of bacterial genomes. Curr Opin Microbiol. Aug 22 2011

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The mechanism by which dsDNA is packaged into tailed bacteriophage virions has fascinated molecular biologists since it was realized, over four decades ago, that these structures contain long dsDNA molecules that are hundreds of times more compact than dsDNA in solution. Experiments in the 1970s showed that, rather than condensing the DNA first and then assembling a shell around this DNA core, tailed-phage DNA is inserted into a preformed protein container (called a prohead or procapsid). Mmolecular genetics studies indicate that the basic machinery of dsDNA packaging is similar in all tailed phages.

The recent burst of structural progress concerning phage DNA-packaging proteins, as well as the introduction of optical tweezer technology into this field, has allowed the formulation of much more detailed ideas for the mechanism by which the DNA-packaging motor pumps DNA into phage procapsids. Potential applications of packaging nanomotors in nanotechnology and biology are beginning to be described, for example these nanomotors could be used for efficient delivery of nucleic acids or related molecules across barriers such as cell membranes or for applications in single-molecule DNA sequencing. There is little doubt that this will be a fertile research area for some time into the future.

The DNA-packaging nanomotor of tailed bacteriophages. 2011 Nat Rev Microbiol. 9(9):647-57 doi: 10.1038/nrmicro2632
Tailed bacteriophages use nanomotors, or molecular machines that convert chemical energy into physical movement of molecules, to insert their double-stranded DNA genomes into virus particles. These viral nanomotors are powered by ATP hydrolysis and pump the DNA into a preformed protein container called a procapsid. As a result, the virions contain very highly compacted chromosomes. Here, I review recent progress in obtaining structural information for virions, procapsids and the individual motor protein components, and discuss single-molecule in vitro packaging reactions, which have yielded important new information about the mechanism by which these powerful molecular machines translocate DNA.

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Researchers have developed a single vaccine which protects against both rabies and Ebola virus. These two viruses are related to each other, but do not cross-react serologically. By inserting elements of the Ebola virus GP protein into an existing rabies virus vaccine, a single bivalent vaccine was produced. Although it works in the laboratory, the new vaccine – or something similar based on this first attempt – need to be tested in primates and eventually in humans.

Apart from people, Ebola virus is thought to have eradicated thousands of gorillas, prompting the World Conservation Union to raise their status to “critically endangered” in 2007, the first time a mammal has become critically endangered as a direct result of disease. Vaccination could help prevent future deaths.

Inactivated or Live-Attenuated Bivalent Vaccines that Confer Protection against Rabies and Ebola Viruses. J Virol. Aug 17 2011
The search for a safe and efficacious vaccine for Ebola virus continues as no current vaccine candidate is nearing licensure. We have developed (a) replication-competent, (b) replication-deficient, and (c) chemically inactivated rabies virus (RABV) vaccines expressing Zaire ebolavirus (ZEBOV) glycoprotein (GP) using a reverse genetics system based on the SAD B19 RABV wildlife vaccine. ZEBOV GP is efficiently expressed by these vaccine candidates and is incorporated into virions. The vaccine candidates were avirulent after inoculation of adult mice, and viruses with a deletion in the RABV glycoprotein have greatly reduced neurovirulence after intracerebral inoculation in suckling mice. Immunization with live or inactivated RABV vaccines expressing ZEBOV GP induced humoral immunity against each virus and conferred protection from both lethal RABV and EBOV challenge in mice. The bivalent RABV/ZEBOV vaccines described here have several distinct advantages that may speed the development of inactivated vaccines for use in humans and potentially live or inactivated vaccines for endemic nonhuman primates at risk of EBOV infection.

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Nanomedicine refers to the medical application of nanotechnology, and particularly to the development of novel nanomaterials that can be used for disease diagnosis and therapy. The unique properties of nanoparticles promise to deliver a new generation of diagnostic reagents with higher signal-to-noise ratios than current imaging modalities, as well as targeted therapies that are more efficacious than today’s medicines and that have fewer adverse effects. Nanomaterials have a large surface-to-volume ratio compared to traditional delivery vehicles that offers a greater capacity for drugs and/or imaging reagents, and the ability to decorate nanoparticles with specific ligands means these diagnostic and therapeutic payloads can be delivered to particular cells. Several classes of nanomaterials are currently being developed, including synthetic materials and naturally occurring bionanomaterials such as viral nanoparticles (VNPs). Each of these systems has benefits and limitations with regard to pharmacokinetics, toxicity, immunogenicity and specificity for the target tissue.

Will virus-based nanoparticles revolutionize medicine or will it all end in grey goo?

Applications of viral nanoparticles in medicine. Curr Opin Biotechnol. May 16 2011
Several nanoparticle platforms are currently being developed for applications in medicine, including both synthetic materials and naturally occurring bionanomaterials such as viral nanoparticles (VNPs) and their genome-free counterparts, virus-like particles (VLPs). A broad range of genetic and chemical engineering methods have been established that allow VNP/VLP formulations to carry large payloads of imaging reagents or drugs. Furthermore, targeted VNPs and VLPs can be generated by including peptide ligands on the particle surface. In this article, we highlight state-of-the-art virus engineering principles and discuss recent advances that bring potential biomedical applications a step closer. Viral nanotechnology has now come of age and it will not be long before these formulations assume a prominent role in the clinic.

The Archaea possess unique metabolic pathways, distinct from those in Bacteria and Eukarya. Based on the genome sequences of the Archaea, there are many cases in which a particular metabolic pathway seems to be absent or incomplete. The search for these ‘missing’ pathways or enzymes has been an exciting field of research in the Archaea. A representative example was the CO₂-fixing mechanisms in autotrophic Crenarchaeota. Although many autotrophic Crenarchaeota had been isolated, homologs of previously recognized CO₂-fixing pathways could not be identified on their genomes. Genes responsible for the degradation and biosynthesis of various sugars had also been unidentified. This paper describes recent findings in archaeal metabolism, including sugar metabolism, CO₂ fixation and a wide range of biosynthetic pathways. The predicted distributions of these pathways, based on genome sequence analyses, in the Archaea are also discussed. These investigations will help understand how microorganisms use and interact with the many natural and man-made compounds they encounter in their environments and also provide the foundation for many biotechnology developments.

Novel metabolic pathways in Archaea, Curr Opin Microbiol. May 23 2011 doi:10.1016/j.mib.2011.04.014
The Archaea harbor many metabolic pathways that differ to previously recognized classical pathways. Glycolysis is carried out by modified versions of the Embden-Meyerhof and Entner-Doudoroff pathways. Thermophilic archaea have recently been found to harbor a bi-functional fructose-1,6-bisphosphate aldolase/phosphatase for gluconeogenesis. A number of novel pentose-degrading pathways have also been recently identified. In terms of anabolic metabolism, a pathway for acetate assimilation, the methylaspartate cycle, and two CO(₂)-fixing pathways, the 3-hydroxypropionate/4-hydroxybutyrate cycle and the dicarboxylate/4-hydroxybutyrate cycle, have been elucidated. As for biosynthetic pathways, recent studies have clarified the enzymes responsible for several steps involved in the biosynthesis of inositol phospholipids, polyamine, coenzyme A, flavin adeninedinucleotide and heme. By examining the presence/absence of homologs of these enzymes on genome sequences, we have found that the majority of these enzymes and pathways are specific to the Archaea.

In this article in Microbiology Today Ed Feil describes how we must brace ourselves for the next wave of data as new sequencing techniques become available to determine and compare many sequences at once. The enormous amount of data soon to be generated will bring exciting new insights into how micro-organisms within communities evolve and interact:

Regardless of the species in question, announcements of completed genome sequencing projects in the mainstream media almost invariably make reference to ‘cracking a code’ or ‘deciphering a genetic blueprint’. For bacteria, these over-used analogies spectacularly fail to give a true sense of the fluidity of genome evolution. The doe-eyed assumption in the mid-1990s that a single genome sequence can safely be considered as a prescriptive ‘solution’ for a given bacterial species has been dramatically falsified. By the late 1990s, multiple genome sequences for Escherichia coli revealed extensive differences in gene content between strains, and it rapidly became clear that, for many taxa, an individual genome is most usefully considered as one of many possible combinations of genes drawn from a vast pool known as the pangenome. When faced with such a maelstrom, our natural inclination (as good cladists) is to try and tidy it up, and catalogue strains into pockets of relatedness. Fortunately, phylogenetic analyses are possible, even for very variable species like E. coli, because one can readily identify genes which are universally present in all strains. These essential ‘core’ genes can be thought of as representing the operating system of a given species. In contrast, the specialist software is provided by ‘non-core’ or ‘accessory’ genes which are variably present or absent, are commonly acquired by horizontal transfer, and tend to be restricted to hypervariable regions called genomic islands. These two sets of genes present a fundamental duality in bacterial genomics. Whilst core genes can satisfy our requirements for molecular phylogeny (i.e. what a strain is), accessory genes often play a significant role in adaptation and phenotypic differences (i.e. what a strain does). Conflicts between these two can go a long way to explaining the mystery behind the muddle that is bacterial systematics.

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Mimivirus, a nucleocytoplasmic large double stranded DNA virus infecting Acanthamoeba species, is the largest virus identified to date. Its icosahedral fibrillated capsid has a diameter of 750 nm. Besides its outstanding particle size, the genome of Mimivirus is also exceptional both in size and complexity. The initial sequencing revealed a linear genome of 1,181,404 nt (roughly the size of the spirochaete bacterium Treponema pallidum genome) harboring 911 protein coding genes and 6 tRNAs. Some of these genes were observed for the first time in a virus, the most salient being those involved in protein translation and DNA repair. These unique features reawaked conceptual discussions on the nature of viruses and the frontier between viruses and cellular organisms.

Breaking the 1000-gene barrier for Mimivirus using ultra-deep genome and transcriptome sequencing. (2011) Virology Journal 2011, 8:99 doi:10.1186/1743-422X-8-99
Background: Mimivirus, a giant dsDNA virus infecting Acanthamoeba, is the prototype of the mimiviridae family, the latest addition to the family of the nucleocytoplasmic large DNA viruses (NCLDVs). Its 1.2 Mb-genome was initially predicted to encode 917 genes. A subsequent RNA-Seq analysis precisely mapped many transcript boundaries and identified 75 new genes.FindingsWe now report a much deeper analysis using the SOLiD technology combining RNA-Seq of the Mimivirus transcriptome during the infectious cycle (202.4 Million reads), and a complete genome re-sequencing (45.3 Million reads). This study corrected the genome sequence and identified several single nucleotide polymorphisms. Our results also provided clear evidence of previously overlooked transcription units, including an important RNA polymerase subunit distantly related to Euryarchea homologues. The total Mimivirus gene count is now 1018, 11% greater than the original annotation. Conclusions: This study highlights the huge progress brought about by ultra-deep sequencing for the comprehensive annotation of virus genomes, opening the door to a complete one-nucleotide resolution level description of their transcriptional activity, and to the realistic modeling of the viral genome expression at the ultimate molecular level. This work also illustrates the need to go beyond bioinformatics-only approaches for the annotation of short protein and non-coding genes in viral genomes.

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