MicrobiologyBytes

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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Human respiratory syncytial virus (HRSV) and human metapneumovirus (HMPV) are common respiratory pathogens. Both viruses comprise two genetic groups, A and B, distinguishable genetically and serologically which circulate with fluctuating frequencies. This gives rise to the observation of switching of the predominantly circulating subtype between seasons. Repeat HRSV infections occur throughout life with decreasing morbidity, and increasingly evidence suggests the same is also true for HMPV. In neither case has it yet been possible to make an effective vaccine against these troublesome pathogens.

Molecular Epidemiology and Evolution of Human Respiratory Syncytial Virus and Human Metapneumovirus. (2011) PLoS ONE 6(3): e17427
Human respiratory syncytial virus (HRSV) and human metapneumovirus (HMPV) are ubiquitous respiratory pathogens of the Pneumovirinae subfamily of the Paramyxoviridae. Two major surface antigens are expressed by both viruses; the highly conserved fusion (F) protein, and the extremely diverse attachment (G) glycoprotein. Both viruses comprise two genetic groups, A and B. Circulation frequencies of the two genetic groups fluctuate for both viruses, giving rise to frequently observed switching of the predominantly circulating group. Nucleotide sequence data for the F and G gene regions of HRSV and HMPV variants from the UK, the Netherlands, Bangkok and data available from Genbank were used to identify clades of both viruses. Several contemporary circulating clades of HRSV and HMPV were identified by phylogenetic reconstructions. The molecular epidemiology and evolutionary dynamics of clades were modelled in parallel. Times of origin were determined and positively selected sites were identified. Sustained circulation of contemporary clades of both viruses for decades and their global dissemination demonstrated that switching of the predominant genetic group did not arise through the emergence of novel lineages each respiratory season, but through the fluctuating circulation frequencies of pre-existing lineages which undergo proliferative and eclipse phases. An abundance of sites were identified as positively selected within the G protein but not the F protein of both viruses. For HRSV, these were discordant with previously identified residues under selection, suggesting the virus can evade immune responses by generating diversity at multiple sites within linear epitopes. For both viruses, different sites were identified as positively selected between genetic groups.

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• Newly discovered respiratory viruses
• Human metapneumovirus glycoprotein G is an important virulence factor

New research is beginning to crack the problem of which strain of flu will be prevalent in a given year, with major implications for global public health preparedness. A computational study of 40 years of flu genomes offers a new way of looking at mutations: by cataloging pairs of genetic changes that have occurred in rapid succession, observing that a mutation in one half of the pair can act as an early warning sign of a mutation about to occur in the other.

Tracking single mutations in a vacuum is not always enough to understand how the flu virus evolves. Sometimes a mutation is functional or adaptive only if it’s in the context of a certain genetic background – that is, if the protein already has some other mutation. The influence such combinations have on an organism’s adaptive fitness is known as epistasis. If you see a mutation occur in Site A and then very soon after you see a mutation in Site B, and this pattern happens repeatedly, then you have some evidence that A and B influence fitness epistatically. The first mutation might be useless on its own, but it might be a prerequisite for the second mutation to be useful. The first mutation is like giving you a nail, and the second one is like giving you a hammer.

Because the studied mutations generally affect the surface proteins that determine whether the virus can enter and infect human cells, being able to predict what mutations are likely to happen in the near future has lifesaving applications. Tens of thousands of Americans, and hundreds of thousands worldwide, die of seasonal flu complications every year. Flu vaccine production is labor intensive and time consuming; to have enough supplies ready for the flu season, public health groups like the Centers for Disease Control and the World Health Organization must make an educated guess as to which strain is likely to be the most active several months in advance. Observing the leading site of an epistatic pair could give them a head start.

Prevalence of Epistasis in the Evolution of Influenza A Surface Proteins. (2011) PLoS Genet 7(2): e1001301. doi:10.1371/journal.pgen.1001301
The surface proteins of human influenza A viruses experience positive selection to escape both human immunity and, more recently, antiviral drug treatments. In bacteria and viruses, immune-escape and drug-resistant phenotypes often appear through a combination of several mutations that have epistatic effects on pathogen fitness. However, the extent and structure of epistasis in influenza viral proteins have not been systematically investigated. Here, we develop a novel statistical method to detect positive epistasis between pairs of sites in a protein, based on the observed temporal patterns of sequence evolution. The method rests on the simple idea that a substitution at one site should rapidly follow a substitution at another site if the sites are positively epistatic. We apply this method to the surface proteins hemagglutinin and neuraminidase of influenza A virus subtypes H3N2 and H1N1. Compared to a non-epistatic null distribution, we detect substantial amounts of epistasis and determine the identities of putatively epistatic pairs of sites. In particular, using sequence data alone, our method identifies epistatic interactions between specific sites in neuraminidase that have recently been demonstrated, in vitro, to confer resistance to the drug oseltamivir; these epistatic interactions are responsible for widespread drug resistance among H1N1 viruses circulating today. This experimental validation demonstrates the predictive power of our method to identify epistatic sites of importance for viral adaptation and public health. We conclude that epistasis plays a large role in shaping the molecular evolution of influenza viruses. In particular, sites with dN=dSv1, which would normally not be identified as positively selected, can facilitate viral adaptation through epistatic interactions with their partner sites. The knowledge of specific interactions among sites in influenza proteins may help us to predict the course of antigenic evolution and, consequently, to select more appropriate vaccines and drugs.

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• Spotting pandemic influenza viruses
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The rotavirus (RV) genome comprises 11 segments of double-stranded RNA (dsRNA) and is contained within a non-enveloped, icosahedral particle. During assembly, a highly coordinated selective packaging mechanism ensures that progeny RV virions contain one of each genome segment. Cis-acting signals thought to mediate assortment and packaging are associated with putative panhandle structures formed by base-pairing of the ends of RV plus-strand RNAs (+RNAs). Viral polymerases within assembling core particles convert the 11 distinct +RNAs to dsRNA genome segments. It remains unclear whether RV +RNAs are assorted before or during encapsidation, and the functions of viral proteins during these processes are not resolved. However, as reviewed in this article, recent insights gained from the study of RV and two other segmented RNA viruses, influenza A virus and bacteriophage Φ6, reveal potential mechanisms of RV assortment and packaging.

Assortment and packaging of the segmented rotavirus genome. Trends Microbiol. Dec 30 2010

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A new study examines how bacterial and archaeal genomic repertoires evolve to face new challenges by acquiring genes from other individuals. Microbes live and thrive in incredibly diverse and harsh conditions, from boiling or freezing water to the human immune system. This remarkable adaptability results from their ability to quickly modify their repertoire of protein functions by gaining, losing and modifying their genes. Microbes were known to modify genes to expand their repertoire of protein families in two ways: via duplication processes followed by slow functional specialization, in the same way as large multicellular organisms like us, and by acquiring different genes directly from other microbes. The latter process, known as horizontal gene transfer (HGT), is notoriously conspicuous in the spread of antibiotic resistance, turning some bacteria into drug-resistant ‘superbugs’ such as MRSA (methicillin-resistant Staphylococcus aureus), a serious public health concern.

The researchers examined a large database of microbial genomes, including some of the most virulent human pathogens, to discover whether duplication or HGT was the most common expansion method. They show that gene family expansion can indeed follow both routes, but unlike large multicellular organisms, it predominantly takes place by horizontal transfer. Thus, quick diversification of microbial functions results from the recruitment by microbes of pre-existing adaptations from other microbes. The study concludes with the observation that, since microbes invented the majority of life’s biochemical diversity, from respiration to photosynthesis, we should recognize the predominant role of HGT in the diversification of all protein families.

Horizontal Transfer, Not Duplication, Drives the Expansion of Protein Families in Prokaryotes. (2011) PLoS Genet 7(1): e1001284. doi:10.1371/journal.pgen.1001284
Gene duplication followed by neo- or sub-functionalization deeply impacts the evolution of protein families and is regarded as the main source of adaptive functional novelty in eukaryotes. While there is ample evidence of adaptive gene duplication in prokaryotes, it is not clear whether duplication outweighs the contribution of horizontal gene transfer in the expansion of protein families. We analyzed closely related prokaryote strains or species with small genomes (Helicobacter, Neisseria, Streptococcus, Sulfolobus), average-sized genomes (Bacillus, Enterobacteriaceae), and large genomes (Pseudomonas, Bradyrhizobiaceae) to untangle the effects of duplication and horizontal transfer. After removing the effects of transposable elements and phages, we show that the vast majority of expansions of protein families are due to transfer, even among large genomes. Transferred genes — xenologs — persist longer in prokaryotic lineages possibly due to a higher/longer adaptive role. On the other hand, duplicated genes — paralogs — are expressed more, and, when persistent, they evolve slower. This suggests that gene transfer and gene duplication have very different roles in shaping the evolution of biological systems: transfer allows the acquisition of new functions and duplication leads to higher gene dosage. Accordingly, we show that paralogs share most protein–protein interactions and genetic regulators, whereas xenologs share very few of them. Prokaryotes invented most of life’s biochemical diversity. Therefore, the study of the evolution of biology systems should explicitly account for the predominant role of horizontal gene transfer in the diversification of protein families.

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• Barriers to Horizontal Gene Transfer
• Evolution of Clostridium difficile

The types of gut bacteria that populate the guts of primates depend on the species of the host as well as where the host lives and what they eat. A new study examines the gut microbial communities in great apes, showing that a host’s species, rather than their diet, has the greatest effect on gut bacteria diversity.

Bacteria are crucial to human health. They enhance the immune system, protect against toxins, and assist in the maturation and renewal of intestinal cells. Gut microbes outnumber our own cells by 10 to 1 but little is known about how certain species come to populate our stomachs, which are sterile at birth. What causes this variation within microbial communities has been a matter of debate. Some scientists have argued that diet and habitat play the most prominent roles. The new research finds that diversity in the composition of these gut communities, not including those occasional transients and unwelcome visitors such as pathogenic bacteria, depends primarily upon the host species.

Using genetic markers, the researchers measured the diversity and abundance of various microbial species found in fecal matter of five great ape species collected in their native ranges and discovered that bacterial populations assorted to species. Moreover, the relationships of the microbial communities matched that of their host. In other words, not only is it possible to differentiate chimpanzees from humans by examining the microbial populations within their guts, but these gut microbes have been tracking the evolution of their hosts for millions of years.

Evolutionary Relationships of Wild Hominids Recapitulated by Gut Microbial Communities. (2010) PLoS Biol 8(11): e1000546. doi:10.1371/journal.pbio.1000546
Multiple factors over the lifetime of an individual, including diet, geography, and physiologic state, will influence the microbial communities within the primate gut. To determine the source of variation in the composition of the microbiota within and among species, we investigated the distal gut microbial communities harbored by great apes, as present in fecal samples recovered within their native ranges. We found that the branching order of host-species phylogenies based on the composition of these microbial communities is completely congruent with the known relationships of the hosts. Although the gut is initially and continuously seeded by bacteria that are acquired from external sources, we establish that over evolutionary timescales, the composition of the gut microbiota among great ape species is phylogenetically conserved and has diverged in a manner consistent with vertical inheritance.

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Several historical epidemic waves of plague have been attributed to Yersinia pestis, the etiologic agent of modern plague. The most famous of these was the second pandemic which was active in Europe from AD 1347 until 1750, and began with the ‘Black Death’. The most informative method to establish the etiological nature of these ancient infections should be the analysis of ancient DNA, but the results of this method have been controversial. By combining ancient DNA analyses and protein-specific detection, this paper demonstrates that Y. pestis caused the Black Death. Furthermore, they show that at least two variants of Y. pestis spread over Europe during the second pandemic. The analysis of up to 20 diagnostic markers reveals that the two variants evolved near the time that phylogenetic branches 1 and 2 separated and may no longer exist. These results resolve a long-standing debate about the etiology of the Black Death and provide key information about the evolution of the plague bacillus and the spread of the disease during the Middle Ages.

Distinct Clones of Yersinia pestis Caused the Black Death. PLoS Pathog 6(10): e1001134. doi:10.1371/journal.ppat.1001134
From AD 1347 to AD 1353, the Black Death killed tens of millions of people in Europe, leaving misery and devastation in its wake, with successive epidemics ravaging the continent until the 18th century. The etiology of this disease has remained highly controversial, ranging from claims based on genetics and the historical descriptions of symptoms that it was caused by Yersinia pestis to conclusions that it must have been caused by other pathogens. It has also been disputed whether plague had the same etiology in northern and southern Europe. Here we identified DNA and protein signatures specific for Y. pestis in human skeletons from mass graves in northern, central and southern Europe that were associated archaeologically with the Black Death and subsequent resurgences. We confirm that Y. pestis caused the Black Death and later epidemics on the entire European continent over the course of four centuries. Furthermore, on the basis of 17 single nucleotide polymorphisms plus the absence of a deletion in glpD gene, our aDNA results identified two previously unknown but related clades of Y. pestis associated with distinct medieval mass graves. These findings suggest that plague was imported to Europe on two or more occasions, each following a distinct route. These two clades are ancestral to modern isolates of Y. pestis biovars Orientalis and Medievalis. Our results clarify the etiology of the Black Death and provide a paradigm for a detailed historical reconstruction of the infection routes followed by this disease.

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• Ancient Plague
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Scientists are studying a retrovirus that has been dormant in chimpanzees and their ancestors for at least one million years. The virus, known as CERV2, is present in the genomes of chimpanzees, but not those of humans, suggesting that chimpanzees’ ancestors became infected after they diverged from human ancestors 5–6 million years ago. The virus was clearly replicating around the time that the first humans were trotting around Africa and beginning to think about colonizing the rest of the world. As a receptor, the ancient virus exploited a transport protein that normally transports copper into and out of cells.

Identification of a receptor for an extinct virus. PNAS USA October 25, 2010 doi: 10.1073/pnas.101234410
The resurrection of endogenous retroviruses from inactive molecular fossils has allowed the investigation of interactions between extinct pathogens and their hosts that occurred millions of years ago. Two such paleoviruses, chimpanzee endogenous retrovirus-1 and -2 (CERV1 and CERV2), are relatives of modern MLVs and are found in the genomes of a variety of Old World primates, but are absent from the human genome. No extant CERV1 and -2 proviruses are known to encode functional proteins. To investigate the host range restriction of these viruses, we attempted to reconstruct functional envelopes by generating consensus genes and proteins. CERV1 and -2 enveloped MLV particles infected cell lines from a range of mammalian species. Using CERV2 Env-pseudotyped MLV reporters, we identified copper transport protein 1 (CTR1) as a receptor that was presumably used by CERV2 during its ancient exogenous replication in primates. Expression of human CTR1 was sufficient to confer CERV2 permissiveness on otherwise resistant hamster cells, and CTR1 knockdown or CuCl2 treatment specifically inhibited CERV2 infection of human cells. Mutations in highly conserved CTR1 residues that have rendered hamster cells resistant to CERV2 include a unique deletion in a copper-binding motif. These CERV2 receptor-inactivating mutations in hamster CTR1 are accompanied by apparently compensating changes, including an increased number of extracellular copper-coordinating residues, and this may represent an evolutionary barrier to the acquisition of CERV2 resistance in primates.

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Biologists have uncovered virus fragments from the same family of the modern Hepatitis B virus locked inside the genomes of songbirds such as the modern-day zebra finch. This article marks the first time that endogenous hepadnaviruses have been found in any organism. An endogenous virus is one that deposits itself or fragments of itself into the chromosome of an organism, allowing it to be passed from generation-to-generation. Previously, most of these known “fossilized” virus sequences have come from retroviruses. These fragments have been sitting in the bird’s genomes for at least 19 million years, far longer than anyone previously thought this family of viruses had been in existence.

The researchers dated the hepadnavirus fragments by locating them in the same spot on the genome of five species of passerine birds and then tracing those species to a common ancestor that lived more than 19 million years ago. This work provides a glimpse into an ancient viral world that we never knew existed. The results are remarkable – hepadnaviruses, and likely many other viruses as well, are far older than we previously thought. Another surprise is that the older versions of the hepadnaviruses are remarkably similar to today’s viruses. This suggests that the slow evolution of the hepadnaviruses observed in birds indicates that the viruses are, in the long run, better adapted to their hosts than what is suggested by study of the disease-causing Hepatitis B viruses. Genomic fossils like these remarkable hepadnaviral fossils have the prospect of completely revising our preconceived notions about the age and evolution of such viruses.

This study also opens new avenues for research that might help predict and prevent human viral pandemics originating in bird species. Given that they were infected in the past, it is legitimate to think that some of these birds may still carry such viruses today. We can use this discovery as a guide to screen targeted groups of bird species for the presence of new circulating Hepatitis B-like viruses.

Genomic Fossils Calibrate the Long-Term Evolution of Hepadnaviruses. (2010) PLoS Biol 8(9): e1000495. doi:10.1371/journal.pbio.1000495
Because most extant viruses mutate rapidly and lack a true fossil record, their deep evolution and long-term substitution rates remain poorly understood. In addition to retroviruses, which rely on chromosomal integration for their replication, many other viruses replicate in the nucleus of their host’s cells and are therefore prone to endogenization, a process that involves integration of viral DNA into the host’s germline genome followed by long-term vertical inheritance. Such endogenous viruses are highly valuable as they provide a molecular fossil record of past viral invasions, which may be used to decipher the origins and long-term evolutionary characteristics of modern pathogenic viruses. Hepadnaviruses (Hepadnaviridae) are a family of small, partially double-stranded DNA viruses that include hepatitis B viruses. Here we report the discovery of endogenous hepadnaviruses in the genome of the zebra finch. We used a combination of cross-species analysis of orthologous insertions, molecular dating, and phylogenetic analyses to demonstrate that hepadnaviruses infiltrated repeatedly the germline genome of passerine birds. We provide evidence that some of the avian hepadnavirus integration events are at least 19 My old, which reveals a much deeper ancestry of Hepadnaviridae than could be inferred based on the coalescence times of modern hepadnaviruses. Furthermore, the remarkable sequence similarity between endogenous and extant avian hepadnaviruses (up to 75% identity) suggests that long-term substitution rates for these viruses are on the order of 10^-8 substitutions per site per year, which is a 1,000-fold slower than short-term rates estimated based on the sequences of circulating hepadnaviruses. Together, these results imply a drastic shift in our understanding of the time scale of hepadnavirus evolution, and suggest that the rapid evolutionary dynamics characterizing modern avian hepadnaviruses do not reflect their mode of evolution on a deep time scale.

Related:

1. Phoenix from the ashes: The 5 million year old virus
2. The Island of Fossil Viruses