MicrobiologyBytes

Grapevines are an important global crop which are widely planted throughout temperate regions. Viruses are a significant factor in reducing the quality and quantity of the yield and are known to reduce the productive life of vineyards. Grapevines are subject to infection by more than 60 different viruses, the most known for any crop plant. Most important grapevine virus diseases are caused by complexes of viruses, with up to nine different viruses having been identified in a single vine. In South Africa, as in most grape-growing regions of the world, grapevine leafroll is regarded to be the most significant virus disease affecting grapevine, with Shiraz disease and Shiraz decline becoming more prominent as emerging diseases in the industry.

Present disease diagnostics rely on ELISA or RT-PCR and target the viruses that have historically been associated with these diseases. While these tests are highly specific, they may not result in an accurate reflection of the etiological status of the tested plant, or of the particular disease, since none of the current diagnostic techniques address the potential contribution of other known or unknown viruses that may be involved in the etiology of a particular disease. Moreover, the error prone replication of RNA viruses leads to quasispecies, which can further complicate PCR-based detection assays as not all variants of the virus may be detected.

New and powerful technologies which are able to sequence viruses from environmental samples without the need for laborious and costly purification, cloning and screening techniques can result in the generation of sequence information for the complete virome in an unbiased fashion. This paper describes the use of sequencing-by-synthesis technology on the massively parallel Illumina Genome Analyzer II, to sequence an environmental sample composed of 44 randomly selected vines, to determine the virus profile of a severely diseased vineyard.

Deep sequencing analysis of viruses infecting grapevines: Virome of a vineyard. Virology. Feb 19 2010
Double stranded RNA, isolated from 44 pooled randomly selected vines from a diseased South African vineyard, has been used in a deep sequencing analysis to build a census of the viral population. The dsRNA was sequenced in an unbiased manner using the sequencing-by-synthesis technology offered by the Illumina Genome Analyzer II and yielded 837 megabases of metagenomic sequence data. Four known viral pathogens were identified. It was found that Grapevine leafroll-associated virus 3 (GLRaV-3) is the most prevalent species, constituting 59% of the total reads, followed by Grapevine rupestris stem pitting-associated virus and Grapevine virus A. Grapevine virus E, a virus not previously reported in South African vineyards, was identified in the census. Viruses not previously identified in grapevine were also detected. The second most prevalent virus detected was a member of the Chrysoviridae family similar to Penicillium chrysogenum virus. Sequences aligning to two other mycoviruses were also detected.

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Biocatalysts are extensively used in the industrial production of bulk chemicals and pharmaceuticals and over 300 processes have been implemented. In the vast majority of processes, enzymes of microbial origin are used as the microbial kingdom represents a huge – and still only partially explored – reservoir for biocatalysts with desired properties. Furthermore, the metagenome approach facilitates the discovery of novel enzymes from microbial sources and hence the number of potentially useful biocatalysts increased exponentially in the past decade.

In contrast, enzymes from animal tissues are less preferred as these often occur as mixture of isoenzymes differing in substrate specificity and product safety (i.e. virus infections), which often restricts their industrial use. Another important source of biocatalysts are plant enzymes. Very often an enzyme does not meet the requirements for a large-scale application and its properties have to be optimized. This usually includes not only the selectivity but also process-related aspects such as long-term stability at certain temperatures or pH-values and activity in the presence of high substrate concentrations to achieve highest productivity.

Beside rather classical strategies such as immobilization, additives or process engineering, molecular biology techniques nowadays represent probably the most important methodology to tailor-design the enzyme for a given process. Two different strategies are used: rational protein design and directed (molecular) evolution, which are increasingly applied in a synergistic manner. Recent advances have mainly focused on applying directed evolution to enzymes, especially important for organic synthesis, such as monooxygenases, ketoreductases, lipases or aldolases in order to improve their activity, enantioselectivity, and stability. The combination of directed evolution and rational protein design using computational tools is becoming increasingly important in order to explore enzyme sequence-space and to create improved or novel enzymes. These developments should allow to further expand the application of microbial enzymes in industry.

Protein engineering of microbial enzymes. Curr Opin Microbiol. Feb 17 2010

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The flavivirus genus includes viruses with a remarkable ability to produce disease on a large scale. The expansion and increased endemicity of dengue and West Nile viruses in the Americas exemplifies their medical and epidemiological importance. The rapid detection of virus infection and induction of the innate antiviral response are crucial to determining the outcome of infection. The intracellular pathogen receptors RIG-I and MDA5 play a central role in detecting flavivirus infections and initiating a robust antiviral response. Yet, these viruses are still capable of producing acute illness in humans. It is now clear that flaviviruses utilize a variety of mechanisms to modulate the interferon response. The non-structural proteins of the various flaviviruses reduce expression of interferon dependent genes by blocking phosphorylation, enhancing degradation or down-regulating expression of major components of the JAK/STAT pathway. Recent studies indicate that interferon modulation is an important factor in the development of severe flaviviral illness. This suggests that an increased understanding of viral-host interactions will facilitate the development of novel therapeutics to treat these viral infections and improved biological models to study flavivirus pathogenesis.

How Flaviviruses Activate and Suppress the Interferon Response. Viruses 2010, 2(2), 676-691; doi:10.3390/v2020676

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Plant diseases are an important constraint on worldwide crop production, accounting for losses of 10–30% of the global harvest each year and represent a significant threat to global food security. Fungal pathogens can broadly be divided into two groups – the biotrophs and necrotrophs. Biotrophic pathogens are parasites that have evolved the means to grow within living plant cells without stimulating plant defence mechanisms. This means that they are able to spread rapidly throughout plant tissue while, at the same time, diverting nutrients from the living plant to fuel their own growth at the expense of plant productivity. In contrast, the necrotrophic pathogens use toxins and depolymerising enzymes to kill and degrade plant cells, consuming the resulting products.

In order to grow, a plant pathogenic fungus must secure an organic carbon source from the plant. In most plant diseases, however, we have little idea of what constitutes the major carbon source for an invading fungus during growth in plant tissue. How do biotrophic plant pathogens acquire nutrients efficiently from a living plant cell? A study published in PLoS Biology provides a significant advance in understanding the mechanism by which a plant pathogenic fungus is able to acquire nutrients in plants. The conclusion is that sucrose, which constitutes the most abundant storage sugar within plants and the product of photosynthesis, is directly utilised by invading pathogens without the need for its extra-cellular degradation by fungal secreted invertases.

A Novel High-Affinity Sucrose Transporter Is Required for Virulence of the Plant Pathogen Ustilago maydis. 2010 PLoS Biol 8(2): e1000303 doi:10.1371/journal.pbio.1000303
The plant parasitic fungus Ustilago maydis is a biotrophic pathogen that depends on live plant tissue for development. It is highly adapted to maize (Zea mays), where it causes the corn smut disease. Fungal cells growing within the plant apoplast are surrounded by the host plasma membrane at all growth stages, thereby establishing tight interaction zones with the host cells that assure optimal access to host-derived nutrients, including organic carbon sources. Here, we focus on the previously unknown feeding mechanisms of this plant pathogen within its host plant. We identified a fungal plasma membrane transporter, Srt1, that is expressed exclusively after plant infection and that turns out to be essential for virulence development of Ustilago in infected plants. Srt1 is the first characterized fungal transporter that allows direct utilization of sucrose without extracellular hydrolysis into monosaccharides, the carbon form more commonly taken up by pathogenic fungi. It is highly specific for sucrose, and its affinity largely exceeds that of equivalent plant transporters. This not only provides advantages for the carbon acquisition by the pathogen, but quite likely also offers a mechanism to prevent induction of plant defense responses known to occur upon apoplastic sucrose hydrolysis.

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Infectious diseases have for centuries ranked with wars and famine as major challenges to human progress and survival. They remain among the leading causes of death and disability worldwide. Against a constant background of established infections, epidemics of new and old infectious diseases periodically emerge, greatly magnifying the global burden of infections. Studies of these emerging infections reveal the evolutionary properties of pathogenic microorganisms and the dynamic relationships between microorganisms, their hosts and the environment.

Emerging infections (EIs) can be defined as “infections that have newly appeared in a population or have existed previously but are rapidly increasing in incidence or geographic range”. EIs have shaped the course of human history and have caused incalculable misery and death. In 1981, a new disease – acquired immune deficiency syndrome (AIDS) – was first recognized. As a global killer, AIDS now threatens to surpass the Black Death of the fourteenth century and the 1918–1920 influenza pandemic, each of which killed at least 50 million people. Of the newly emerging and re-emerging/resurging diseases that have followed the appearance of AIDS, some have been minor curiosities, such as the 2003 cases of monkeypox imported into the United States, whereas others, such as severe acute respiratory syndrome (SARS), which emerged in the same year, have had a worldwide impact. The 2001 anthrax bioterrorist attack in the United States falls into a third category: deliberately emerging diseases. EIs can be expected to remain a considerable challenge for the foreseeable future. Emergence results from dynamic interactions between rapidly evolving infectious agents and changes in the environment and in host behaviour that provide such agents with favourable new ecological niches. This review examines the nature and scope of emerging and re-emerging microbial threats and considers methods for their control.

The challenge of emerging and re-emerging infectious diseases. Nature 430, 242-249, 2004 doi:10.1038/nature02759

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Viruses have represented a constant threat to human communities throughout their history, and human genes involved in anti-viral responses are targets of virus-driven selective pressure. A new study utilizes the signs left by genetic selection to identify virus infection-associated allelic variants. By analysing more than 660,000 single nucleotide polymorphisms (SNPs) in 52 human populations, the study uses virus diversity (the number of different viruses in a geographic region) to measure virus-driven selective pressure. The results showed that genes involved in the immune response and in the biosynthesis of glycan structures functioning as virus receptors display more variants associated with virus diversity than expected by chance. The same holds true for genes encoding proteins that directly interact with virus components. Genome-wide analysis identified 441 variants mapping to 139 human genes significantly associated with virus diversity. The authors analyzed the functional relationships among genes subjected to virus-driven selective pressure and identified a complex interaction network enriched in viral products-interacting proteins. This is a novel approach to the identification of gene variants that may be involved in the susceptibility to virus infections.

Genome-Wide Identification of Susceptibility Alleles for Viral Infections through a Population Genetics Approach. 2010 PLoS Genet 6(2): e1000849. doi:10.1371/journal.pgen.1000849
Viruses have exerted a constant and potent selective pressure on human genes throughout evolution. We utilized the marks left by selection on allele frequency to identify viral infection-associated allelic variants. Virus diversity (the number of different viruses in a geographic region) was used to measure virus-driven selective pressure. Results showed an excess of variants correlated with virus diversity in genes involved in immune response and in the biosynthesis of glycan structures functioning as viral receptors; a significantly higher than expected number of variants was also seen in genes encoding proteins that directly interact with viral components. Genome-wide analyses identified 441 variants significantly associated with virus-diversity; these are more frequently located within gene regions than expected, and they map to 139 human genes. Analysis of functional relationships among genes subjected to virus-driven selective pressure identified a complex network enriched in viral products-interacting proteins. The novel approach to the study of infectious disease epidemiology presented herein may represent an alternative to classic genome-wide association studies and provides a large set of candidate susceptibility variants for viral infections.

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Noroviruses are the most common cause of food-borne gastroenteritis worldwide, and explosive outbreaks frequently occur in community settings, where the virus can immobilize large numbers of infected individuals for 24–48 hours, making the development of effective vaccines and antiviral therapies a priority. There are currently no vaccines or antiviral treatments available to treat or prevent the >260 million gastroenteritis cases reported globally each year. Noroviruses have proven difficult to work with in the laboratory owing to the lack of cell culture systems and animal models, and therefore little is known about the pathogenesis caused by this virus, which has hampered the development of efficacious therapeutics.

Several challenges have hampered therapeutic design, including: the limitations of cell culture and small-animal model systems; the complex effects of host pre-exposure histories; differential host susceptibility, which is correlated with blood group and secretor status; and the evolution of novel immune escape variants. This review discusses the molecular and structural mechanisms that facilitate the persistence of noroviruses in human populations.

Viral shape-shifting: norovirus evasion of the human immune system. 2010 Nature Reviews Microbiology 8, 231-241 doi:10.1038/nrmicro2296

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The first production from an oil well is the result of the pressure either of the pressure of earth’s overburden on the oil-bearing formation, or by pumping to create negative pressure. As this primary production declines, some oil wells are converted to injector wells, and either waterflooding or sometimes gas flooding are implemented. Even after this secondary production effort has reached its economic limit, two-thirds of the original oil in place is still left in the ground and tertiary measures may be employed. These include chemical enhanced oil recovery (EOR) methods such as polymer flooding, surfactant flooding, alkaline flooding, etc. or the use of thermal measures such as injection of steam or in situ combustion.

Another tertiary method of oil recovery is microbial enhanced oil recovery, commonly referred to as MEOR. There are several ways in which microorganisms can enhance oil recovery other than what is commonly referred to as MEOR. Microorganisms can be used to reduce the paraffin build-up in producing wells or they can be utilized to produce solvents or polymers above ground for pumping into the oil-bearing formation as in EOR. In reality, the difference between EOR and some of the MEOR methods is the means by which the recovery-enhancing chemicals are introduced into the reservoir. Normally however, MEOR refers to the use of microorganisms in the oil-bearing formation itself to enhance oil recovery.

Since 1946 more than 400 patents on MEOR have been issued, but none has gained acceptance by the oil industry. Most of the literature on MEOR is from laboratory experiments or from field trials of insufficient duration or that lack convincing proof of the process. Several authors have made recommendations required to establish MEOR as a viable method to enhance oil recovery, and until these tests are performed, MEOR will remain an unproven concept rather than a highly desirable reality.

Microbial enhanced oil recovery (MEOR). Curr Opin Microbiol. Feb 8 2010

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Influenza A virus is the prototype of the Orthomyxoviridae, and like all members of this family, the negative-sense RNA that comprises its genome is divided into separate segments. These vRNA segments share a common organisation; a long central coding region (in antisense), sometimes encoding more than one polypeptide, flanked by relatively short untranslated regions (UTRs) and at the termini, sequences conserved between segments that show partial complementarity. The vRNA segments are separately encapsidated into ribonucleoprotein (RNP) structures by viral polypeptides. These RNPs act as independent units for the purposes of viral RNA synthesis, which occurs in the nuclei of infected cells. Replicated vRNAs are exported (as RNPs) from the nucleus via the cellular CRM1 pathway, and at the final stage of viral assembly, are incorporated into the virion as it buds from the apical plasma membrane of the cell. The process of virion assembly is not well understood but is thought to involve a series of protein-protein interactions between the cytoplasmic tails of the viral integral membrane proteins, the matrix protein and the RNPs.

Genome segmentation confers evolutionary advantages on influenza viruses, but also poses a problem in virion assembly. The eight segments encode 12 identified polypeptides. At least one copy of each of the eight vRNAs must be packaged for a single virion to be able to initiate a productive infection. Until recently, the process by which this was achieved was poorly understood, but a clearer picture has begun to emerge of a mechanism for specifically packaging a full genome, mediated by cis-acting packaging signals in the vRNAs. This review aims to summarise the thought processes and experimental evidence leading up to the currently accepted model for influenza A genome packaging and to highlight the main questions remaining.

Genome packaging in influenza A virus. J Gen Virol. Dec 2 2009

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