MicrobiologyBytes

While single cell studies have focused on bacteria and cyanobacteria, single virions have yet to be isolated and genomically described using similar mechanisms. Viruses are ubiquitous and the most numerous and diverse biological entities on our planet. Nearly all aspects of our lives are influenced by viruses through shaping the environments that surround us, our immune responses and even our genomes. The field of environmental viral metagenomics has gained momentum over the past several years; however, sequencing of individual environmental viral genomes is currently dependent on the establishment of cultivable virus-host systems. With this in mind, if less than one percent of microbial populations can be cultured using standard microbiological techniques due to incongruencies in direct counts versus cultivatable microbes, then only a very small number of viruses have the likelihood of being genomically described. Currently, viral genomic sequences are lacking in public databases, with the exception of human viruses and those of agricultural and industrial significance (e.g. Lactococcal phages). Clearly, a better understanding of virus diversity and evolution will not be achieved until the genomes of a broad range of viruses are available.

This paper introduces an approach for isolating and characterizing the genomes of viruses called “Single Virus Genomics” (SVG). The benefits of SVG will be far-reaching, enabling novel virus discovery in a variety of clinical and environmental settings, altering our understanding of virus evolution, adaptation and ecology and facilitating the interpretation of viral genomic and metagenomic data by providing suitable reference genomes.

Single Virus Genomics: A New Tool for Virus Discovery. 2011 PLoS ONE 6(3): e17722. doi:10.1371/journal.pone.0017722
Whole genome amplification and sequencing of single microbial cells has significantly influenced genomics and microbial ecology by facilitating direct recovery of reference genome data. However, viral genomics continues to suffer due to difficulties related to the isolation and characterization of uncultivated viruses. We report here on a new approach called ‘Single Virus Genomics’, which enabled the isolation and complete genome sequencing of the first single virus particle. A mixed assemblage comprised of two known viruses; E. coli bacteriophages lambda and T4, were sorted using flow cytometric methods and subsequently immobilized in an agarose matrix. Genome amplification was then achieved in situ via multiple displacement amplification (MDA). The complete lambda phage genome was recovered with an average depth of coverage of approximately 437X. The isolation and genome sequencing of uncultivated viruses using Single Virus Genomics approaches will enable researchers to address questions about viral diversity, evolution, adaptation and ecology that were previously unattainable.

Since ancient times, Yersinia pestis has wreaked havoc on the human population. In this article in Microbiology Today (pdf) Petra Oyston asks what can the transmission and evolution of this unusual pathogen teach us about how we might prepare for future emergent pathogens?

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In October 2009 it was reported that 68 of 101 patients with chronic fatigue syndrome (CFS) in the USA were infected with a novel gamma retrovirus, xenotropic murine leukaemia virus-related virus (XMRV), a virus previously linked to prostate cancer. This finding, if confirmed, would have a profound effect on the understanding and treatment of an incapacitating disease affecting millions worldwide.

But does XMRV cause disease? It’s far from clear that it does.

No Evidence of XMRV or Related Retroviruses in a London HIV-1-Positive Patient Cohort. 2011 PLoS ONE 6(3): e18096. doi:10.1371/journal.pone.0018096
Background
Several studies have implicated a recently discovered gammaretrovirus, XMRV (Xenotropic murine leukaemia virus-related virus), in chronic fatigue syndrome and prostate cancer, though whether as causative agent or opportunistic infection is unclear. It has also been suggested that the virus can be found circulating amongst the general population. The discovery has been controversial, with conflicting results from attempts to reproduce the original studies.
Methodology/Principal Findings
We extracted peripheral blood DNA from a cohort of 540 HIV-1-positive patients (approximately 20% of whom have never been on anti-retroviral treatment) and determined the presence of XMRV and related viruses using TaqMan PCR. While we were able to amplify as few as 5 copies of positive control DNA, we did not find any positive samples in the patient cohort.
Conclusions/Significance
In view of these negative findings in this highly susceptible group, we conclude that it is unlikely that XMRV or related viruses are circulating at a significant level, if at all, in HIV-1-positive patients in London or in the general population.

Ground zero for vaccinology is Edward Jenner’s home in Berkeley, England. Here Edward Jenner worked, studied, and practiced as a country doctor – and later dominated a decade of my life! Jenner knew that poets talked about the nice complexions of milkmaids, and he heard a milkmaid say she was immune to smallpox because she had acquired cowpox. He came to believe in the protective effects of cowpox after careful review of the experiences of milkmaids during smallpox outbreaks. He spent a dozen years observing before he experimented with the transfer of cowpox from a lesion on the hand of Sarah Nelms, a milkmaid, to 8-year-old James Phipps, the son of a local laborer, in May 1796. Although Jenner had no concept of viruses, immune systems, or vaccinology, he used science to help imitate what he saw happening in nature.

I visited the museum several years ago and was impressed. If you ever have the chance – go there.

Edward Jenner Museum. Emerg Infect Dis Volume 17, Number 4–April 2011 doi: 10.3201/eid1704.101680

Related:

• A dose of the pox: An interactive tutorial that steps you through the discovery & ultimate eradication of smallpox using a series of questions. At each step, information & images are provided to help broaden ones appreciation of this scientific feat.

I have recently started my contributions to the OeRBITAL project. Over the next few weeks I’ll be adding my contributions to the Microbiology page. The OeRBITAL wiki is not open for public contributions, but I am calling here for your suggestions of good open educational resources in microbiology, so comments below please!

Recognized as a global problem, antibiotic resistance increases the morbidity and mortality caused by bacterial infections, as well as the cost of treating infectious diseases. The threat from resistance (particularly multiple resistance in bacterial strains that are widely disseminated) is serious. The key factors contributing to this threat are the pressure of increased antibiotic usage (in both human and animal medicine), greater mobility of the population and industrialization. Many potentially life-threatening infections, generally regarded as diseases from the past due to the success of antibiotics and vaccines, have returned as resistance increasingly hampers successful therapy and prophylaxis.

Several studies have focused on antibiotic resistance codification in plasmids or transposons, and there is also interesting information about the extent of antibiotic resistance genes in a given environment (the so-called “resistome”). However, there is less information on the potential contribution of phages to antibiotic resistance-gene transfer, despite calls for research in this field. Recent reports conclude that the horizontal transfer of genetic information by phages is much more prevalent than previously thought, and that the environment plays a crucial role in the phage-mediated transfer of antibiotic-resistance genes. This paper highlights the potential role of phages in the spread of these genes in the aquatic environment.

Antibiotic Resistance Genes in the Bacteriophage DNA Fraction of Environmental Samples. (2011) PLoS ONE 6(3): e17549. doi:10.1371/journal.pone.0017549
Antibiotic resistance is an increasing global problem resulting from the pressure of antibiotic usage, greater mobility of the population, and industrialization. Many antibiotic resistance genes are believed to have originated in microorganisms in the environment, and to have been transferred to other bacteria through mobile genetic elements. Among others, β-lactam antibiotics show clinical efficacy and low toxicity, and they are thus widely used as antimicrobials. Resistance to β-lactam antibiotics is conferred by β-lactamase genes and penicillin-binding proteins, which are chromosomal- or plasmid-encoded, although there is little information available on the contribution of other mobile genetic elements, such as phages. This study is focused on three genes that confer resistance to β-lactam antibiotics, namely two β-lactamase genes (blaTEM and blaCTX-M9) and one encoding a penicillin-binding protein (mecA) in bacteriophage DNA isolated from environmental water samples. The three genes were quantified in the DNA isolated from bacteriophages collected from 30 urban sewage and river water samples, using quantitative PCR amplification. All three genes were detected in the DNA of phages from all the samples tested, in some cases reaching 104 gene copies (GC) of blaTEM or 102 GC of blaCTX-M and mecA. These values are consistent with the amount of fecal pollution in the sample, except for mecA, which showed a higher number of copies in river water samples than in urban sewage. The bla genes from phage DNA were transferred by electroporation to sensitive host bacteria, which became resistant to ampicillin. blaTEM and blaCTX were detected in the DNA of the resistant clones after transfection. This study indicates that phages are reservoirs of resistance genes in the environment.

“New diagnoses for people infected with HIV in the UK almost doubled over the past decade, (from 1,950 in 2001 to 3,780 in 2010) according to new figures released today by the Health Protection Agency (HPA). If these 3,780 UK-acquired HIV cases in 2010 had been prevented, over £32 million annually or £1.2 billion over a lifetime in costs would have been saved. Men who have sex with men remain the group most at risk of becoming infected with HIV in the UK and new diagnoses in this group alone have increased by 70 per cent in the past 10 years rising from 1,810 in 2001 to 3,080 in 2010. Late diagnosis continues to severely affect the health outcomes of people with HIV. On average, of all those who die from HIV infection every year, three out of five are diagnosed late – that is after the point their treatment should have begun. New guidance released today by the National Institute of Clinical Excellence recommends increased testing of HIV in key risk groups. In the UK black Africans and men who have sex with men are most at risk of becoming infected with HIV. Increased testing will encourage early diagnosis in these groups.”

via HPA – UK-acquired HIV nearly doubles in ten years

How proteins and nucleic acids assemble, often spontaneously, into structurally well-defined three-dimensional objects is an intriguing question. The limited size of the phage genome and the multicomponent composition of bacteriophages make them well suited for assembly investigations. Genetic manipulation of phages has made it easy to observe the effects of gene inactivation on protein-protein association, providing information on the sequence of assembly processes. Over the past fifty years, mutational, biochemical and biophysical analyses, X-ray crystallography, NMR, cryo-electron microscopy (cryo-EM), thin sectioning and single molecule methods have been used to study bacteriophages. This review describes what has been achieved and contemplates what still needs to be accomplished, focusing mostly on dsDNA tailed phages.

Bacteriophage Assembly. Viruses 2011, 3(3), 172-203; doi:10.3390/v3030172
Bacteriophages have been a model system to study assembly processes for over half a century. Formation of infectious phage particles involves specific protein-protein and protein-nucleic acid interactions, as well as large conformational changes of assembly precursors. The sequence and molecular mechanisms of phage assembly have been elucidated by a variety of methods. Differences and similarities of assembly processes in several different groups of bacteriophages are discussed in this review. The general principles of phage assembly are applicable to many macromolecular complexes.

It is well established that the genomes of many dsDNA tailed bacteriophages have mosaic relationships, where the mosaicism is defined as patchy sequence similarity when two related phage genomes are compared. The regions with different extents of similarity have been interpreted to be genome sections with different evolutionary histories that have been horizontally exchanged among phages. Such exchangeable genome segments have been called “modules”, and these modules are hypothesized to be minimal autonomously functional units, such as groups of genes that must function together or single proteins or even protein domains that function independently. Phage genome mosaicism can range from quantitative differences in the extent of sequence similarity between homologous regions to parallel non-homologous genome sections that encode completely different proteins.

A quantitative understanding of the extant diversity, exchange rates and precise boundaries of these genetic “modules” has remained elusive. Recent advances in the ease of DNA sequence determination have resulted in a rapid increase in the number of phage genome sequences available, and these provide an opportunity for much more detailed and robust analyses of phage genome mosaicism. This paper focuses on the virion assembly genes of fifty-seven different of phages that are “closely” related to Salmonella enterica phage P22. Analysis of such a large number of unambiguously orthologous gene sets is powerful, and analysis of phage P22 is particularly informative because of the extensive body of experimental work on phage P22 virion structure and the specific roles and interactions of its morphogenetic proteins.

Evolution of mosaically related tailed bacteriophage genomes seen through the lens of phage P22 virion assembly. Virology. 2011 411(2): 393-415
The mosaic composition of the genomes of dsDNA tailed bacteriophages (Caudovirales) is well known. Observations of this mosaicism have generally come from comparisons of small numbers of often rather distantly related phages, and little is known about the frequency or detailed nature of the processes that generate this kind of diversity. Here we review and examine the mosaicism within fifty-seven clusters of virion assembly genes from bacteriophage P22 and its “close” relatives. We compare these orthologous gene clusters, discuss their surprising diversity and document horizontal exchange of genetic information between subgroups of the P22-like phages as well as between these phages and other phage types. We also point out apparent restrictions in the locations of mosaic sequence boundaries in this gene cluster. The relatively large sample size and the fact that phage P22 virion structure and assembly are exceptionally well understood make the conclusions especially informative and convincing.