MicrobiologyBytes

I make no secret of my admiration for Mimivirus, the largest virus known. I make quite a play of what Mimivirus tells us about the nature of viruses and virus evolution in the new edition of Principles of Molecular Virology:

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But of course, as soon as you publish a printed textbook, it’s out of date (that’s why I write this blog). And so it proved this week when Mimivirus was knocked off it’s throne by the latest Girus to come along – the even bigger OMGItsSoHugeItBlocksOutTheSun virus. Well, actually, they called it Megavirus (but you get the general idea). With a genome of 1.26 million base pairs of DNA (megabases), this is now the largest virus known (until we discover an even bigger one).

So what do these monsters tell us about viruses? Probably quite a lot, as this excellent Wired article describes.

See:
Distant Mimivirus relative with a larger genome highlights the fundamental features of Megaviridae. PNAS USA 10 Oct 2011. DOI: 10.1073/pnas.1110889108
Mimivirus, a DNA virus infecting acanthamoeba, was for a long time the largest known virus both in terms of particle size and gene content. Its genome encodes 979 proteins, including the first four aminoacyl tRNA synthetases (ArgRS, CysRS, MetRS, and TyrRS) ever found outside of cellular organisms. The discovery that Mimivirus encoded trademark cellular functions prompted a wealth of theoretical studies revisiting the concept of virus and associated large DNA viruses with the emergence of early eukaryotes. However, the evolutionary significance of these unique features remained impossible to assess in absence of a Mimivirus relative exhibiting a suitable evolutionary divergence. Here, we present Megavirus chilensis, a giant virus isolated off the coast of Chile, but capable of replicating in fresh water acanthamoeba. Its 1,259,197-bp genome is the largest viral genome fully sequenced so far. It encodes 1,120 putative proteins, of which 258 (23%) have no Mimivirus homologs. The 594 Megavirus/Mimivirus orthologs share an average of 50% of identical residues. Despite this divergence, Megavirus retained all of the genomic features characteristic of Mimivirus, including its cellular-like genes. Moreover, Megavirus exhibits three additional aminoacyl-tRNA synthetase genes (IleRS, TrpRS, and AsnRS) adding strong support to the previous suggestion that the Mimivirus/Megavirus lineage evolved from an ancestral cellular genome by reductive evolution. The main differences in gene content between Mimivirus and Megavirus genomes are due to (i) lineages specific gains or losses of genes, (ii) lineage specific gene family expansion or deletion, and (iii) the insertion/migration of mobile elements (intron, intein).

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Two days ago I wrote about monkeys as an animal model for smallpox. Animal models certainly have a place in microbiology research, but here’s a rather less controversial one:

Pseudomonas aeruginosa causes serious infections in people with compromised immune systems. Individuals with cystic fibrosis and hospital patients are particularly vulnerable to P. aeruginosa infections. This bacterium does not respond to many antibiotics, making these infections difficult to treat. P. aeruginosa can grow as free-floating planktonic cells or as microcolonies known as biofilms. The ability of P. aeruginosa to form biofilms is thought to contribute to their ability to cause chronic infections. The aim of this research was to develop a simple biofilm model of infection using the fruit fly (Drosophila melanogaster). The immune system of the fruit fly has similarities with the vertebrate innate immune system. Understanding how P. aeruginosa causes infections in Drosophila will aid in understanding virulence mechanisms in mammals. This study shows that feeding P. aeruginosa to Drosophila results in a biofilm infection and biofilm infections induced expression of antimicrobial peptide immune response genes in the fly. Using fly survival as a measure of virulence it shows that biofilm infections were less virulent than non-biofilm infections. These results provide novel insight into host-pathogens interactions during P. aeruginosa infection.

Drosophila melanogaster as an Animal Model for the Study of Pseudomonas aeruginosa Biofilm Infections In Vivo. (2010) PLoS Pathog 7(10): e1002299. doi:10.1371/journal.ppat.1002299
Pseudomonas aeruginosa is an opportunistic pathogen capable of causing both acute and chronic infections in susceptible hosts. Chronic P. aeruginosa infections are thought to be caused by bacterial biofilms. Biofilms are highly structured, multicellular, microbial communities encased in an extracellular matrix that enable long-term survival in the host. The aim of this research was to develop an animal model that would allow an in vivo study of P. aeruginosa biofilm infections in a Drosophila melanogaster host. At 24 h post oral infection of Drosophila, P. aeruginosa biofilms localized to and were visualized in dissected Drosophila crops. These biofilms had a characteristic aggregate structure and an extracellular matrix composed of DNA and exopolysaccharide. P. aeruginosa cells recovered from in vivo grown biofilms had increased antibiotic resistance relative to planktonically grown cells. In vivo, biofilm formation was dependent on expression of the pel exopolysaccharide genes, as a pelB::lux mutant failed to form biofilms. The pelB::lux mutant was significantly more virulent than PAO1, while a hyperbiofilm strain (PAZHI3) demonstrated significantly less virulence than PAO1, as indicated by survival of infected flies at day 14 postinfection. Biofilm formation, by strains PAO1 and PAZHI3, in the crop was associated with induction of diptericin, cecropin A1 and drosomycin antimicrobial peptide gene expression 24 h postinfection. In contrast, infection with the non-biofilm forming strain pelB::lux resulted in decreased AMP gene expression in the fly. In summary, these results provide novel insights into host-pathogen interactions during P. aeruginosa oral infection of Drosophila and highlight the use of Drosophila as an infection model that permits the study of P. aeruginosa biofilms in vivo.

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It seems like only yesterday we were on the verge of international agreement to destroy all remaing laboratory stocks of smallpox (variola) virus. So it comes as something of a surprise to find people infecting monkeys with smallpox to study the pathogenesis of the disease. Such studies significantly advance our understanding of variola pathogenesis in primates and could help development of new antiviral drugs, improved bioterrorism countermeasures, and suggest new potential targets for therapeutic intervention in humans.

But is it a good idea?

Progression of Pathogenic Events in Cynomolgus Macaques Infected with Variola Virus. (2011) PLoS ONE 6(10): e24832. doi:10.1371/journal.pone.0024832
Smallpox, caused by variola virus (VARV), is a devastating human disease that affected millions worldwide until the virus was eradicated in the 1970 s. Subsequent cessation of vaccination has resulted in an immunologically naive human population that would be at risk should VARV be used as an agent of bioterrorism. The development of antivirals and improved vaccines to counter this threat would be facilitated by the development of animal models using authentic VARV. Towards this end, cynomolgus macaques were identified as adequate hosts for VARV, developing ordinary or hemorrhagic smallpox in a dose-dependent fashion. To further refine this model, we performed a serial sampling study on macaques exposed to doses of VARV strain Harper calibrated to induce ordinary or hemorrhagic disease. Several key differences were noted between these models. In the ordinary smallpox model, lymphoid and myeloid hyperplasias were consistently found whereas lymphocytolysis and hematopoietic necrosis developed in hemorrhagic smallpox. Viral antigen accumulation, as assessed immunohistochemically, was mild and transient in the ordinary smallpox model. In contrast, in the hemorrhagic model antigen distribution was widespread and included tissues and cells not involved in the ordinary model. Hemorrhagic smallpox developed only in the presence of secondary bacterial infections – an observation also commonly noted in historical reports of human smallpox. Together, our results support the macaque model as an excellent surrogate for human smallpox in terms of disease onset, acute disease course, and gross and histopathological lesions.

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Bacteriophage lambda is a model phage for most other dsDNA phages and has been studied for over 60 years. Although it is probably the best-characterized phage there are still about 20 poorly understood open reading frames in its 48-kb genome. For a complete understanding we need to know all interactions among its proteins. A new paper has examined the lambda literature and compiled a total of 33 interactions that have been found among lambda proteins. The authors set out to find out how many protein-protein interactions remain to be found in this phage.

In order to map lambda’s interactions, they cloned 68 out of 73 lambda open reading frames (the “ORFeome”) into Gateway vectors and systematically tested all proteins for interactions using exhaustive array-based yeast two-hybrid screens. These screens identified 97 interactions, including 16 out of 30 previously published interactions (53%). They also also found at least 18 new plausible interactions among functionally related proteins. All previously found and new interactions are combined into structural and network models of phage lambda.

Phage lambda serves as a benchmark for future studies of protein interactions among phage, viruses in general, or large protein assemblies. We conclude that we could not find all the known interactions because they require chaperones, post-translational modifications, or multiple proteins for their interactions. The lambda protein network connects 12 proteins of unknown function with well characterized proteins, which should shed light on the functional associations of these uncharacterized proteins.

The protein interaction map of bacteriophage lambda. BMC Microbiology 2011, 11:213 doi:10.1186/1471-2180-11-213

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Flagella-driven bacterial motility, referred to as swimming, has been recognized for over 20 years to affect the ability of bacteria to infect and colonize a host. The common theme is that bacteria must be motile to colonize the host but must become non-motile to chronically persist; this has been observed in many pathogenic bacteria including species of Vibrio and Pseudomonas. Therefore it makes sense that the immune system would evolve mechanisms to exploit this virulence determinant of pathogenic bacteria. This paper presents evidence that flagellar motility is recognized by innate immune cells as a phagocytic activation signal. It shows that step-wise loss of flagellar motility confers a proportional ability to evade phagocytic engulfment, independent of the flagellum itself acting as a phagocytic activator. This is not due to motility- co-regulated secretions or compensatory genetic changes by the bacteria, but instead is due to a mechano-sensory response whereby phagocytic cells respond directly to flagellar motility. This represents a novel mechanism by which the innate immune system facilitates clearance of bacterial pathogens, and provides an explanation for how selective pressure may result in bacteria with down-regulated flagellar gene expression and motility as is observed in isolates taken from chronic infections.

Step-Wise Loss of Bacterial Flagellar Torsion Confers Progressive Phagocytic Evasion. (2011) PLoS Pathog 7(9): e1002253. doi:10.1371/journal.ppat.1002253
Phagocytosis of bacteria by innate immune cells is a primary method of bacterial clearance during infection. However, the mechanisms by which the host cell recognizes bacteria and consequentially initiates phagocytosis are largely unclear. Previous studies of the bacterium Pseudomonas aeruginosa have indicated that bacterial flagella and flagellar motility play an important role in colonization of the host and, importantly, that loss of flagellar motility enables phagocytic evasion. Here we use molecular, cellular, and genetic methods to provide the first formal evidence that phagocytic cells recognize bacterial motility rather than flagella and initiate phagocytosis in response to this motility. We demonstrate that deletion of genes coding for the flagellar stator complex, which results in non-swimming bacteria that retain an initial flagellar structure, confers resistance to phagocytic binding and ingestion in several species of the gamma proteobacterial group of Gram-negative bacteria, indicative of a shared strategy for phagocytic evasion. Furthermore, we show for the first time that susceptibility to phagocytosis in swimming bacteria is proportional to mot gene function and, consequently, flagellar rotation since complementary genetically- and biochemically-modulated incremental decreases in flagellar motility result in corresponding and proportional phagocytic evasion. These findings identify that phagocytic cells respond to flagellar movement, which represents a novel mechanism for non-opsonized phagocytic recognition of pathogenic bacteria.

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As a new paper confirms the presence of thousands of “unknown” viruses in raw sewage, it brings to mind something we already knew – that although there are a lot of “unknown” viruses out there, most of them are tailed bacteriophages, blown apart, reshuffled and reassembled:

Raw Sewage Harbors Diverse Viral Populations. mBio 2 (5) e00180-11 4 October 2011 doi: 10.1128/​mBio.00180-11
At this time, about 3,000 different viruses are recognized, but metagenomic studies suggest that these viruses are a small fraction of the viruses that exist in nature. We have explored viral diversity by deep sequencing nucleic acids obtained from virion populations enriched from raw sewage. We identified 234 known viruses, including 17 that infect humans. Plant, insect, and algal viruses as well as bacteriophages were also present. These viruses represented 26 taxonomic families and included viruses with single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), positive-sense ssRNA [ssRNA(+)], and dsRNA genomes. Novel viruses that could be placed in specific taxa represented 51 different families, making untreated wastewater the most diverse viral metagenome (genetic material recovered directly from environmental samples) examined thus far. However, the vast majority of sequence reads bore little or no sequence relation to known viruses and thus could not be placed into specific taxa. These results show that the vast majority of the viruses on Earth have not yet been characterized. Untreated wastewater provides a rich matrix for identifying novel viruses and for studying virus diversity.

Analysis of the virus population present in equine faeces indicates the presence of hundreds of uncharacterized virus genomes. (2005) Virus Genes. 30(2): 151-156
Virus DNA was isolated from horse faeces and cloned in a sequence-independent fashion. 268 clones were sequenced and 178140 nucleotides of sequence obtained. Statistical analysis suggests the library contains 17560 distinct clones derived from up to 233 different virus genomes. TBLASTX analysis showed that 32% of the clones had significant identity to GenBank entries. Of these 63% were viral; 20% bacterial; 7% archaeal; 6% eukarya; and 5% were related to mobile genetic elements. Fifty-two percent of the virus identities were with Siphoviridae; 26% unclassified phages; 17% Myoviridae; 4% Podoviridae; and one clone (2%) was a vertebrate Orthopoxvirus. Genes coding for predicted virus structural proteins, proteases, glycosidases and nucleic acid-binding proteins were common.

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Hendra virus (HeV) belongs to the genus Henipavirus (family Paramyxoviridae), and is an emerging zoonotic virus. The virus is transmitted to humans via an intermediary equine host from bats of the genus Pteropus, colloquially referred to as flying foxes. HeV was first identified in 1994 following an outbreak in Hendra, a suburb of Brisbane, Queensland, Australia that resulted in the infection of 20 horses and two humans. There have been 31 identified spillovers of Hendra virus, resulting in a total 66 attributed equine cases and 7 human cases resulting in 4 human deaths. In an unprecedented year for HeV activity, 17 spillovers resulting in 21 infections in horses have been identified between June and August 2011. The first infection in a dog was also diagnosed. Due to its wide host range, high mortality and lack of effective prevention or treatment modalities, HeV is classified in the highest biological safety category – BSL4.

Multiple genetic variants of HeV circulating at one time were observed previously in July 2008 when there were two concurrent outbreaks in horses over 930 km apart. Spillovers of HeV from flying foxes into horses are most likely due to the increased incidence of horses coming into contact with excretions from flying foxes when compared to humans. In addition, horses may be more susceptible to HeV infection as their innate immune response genes are genetically most closely related to flying foxes. Intimate contact between horses and humans have been found to be required for infection, however, no direct transmission from bats to humans has been detected. The possibility of direct HeV infection from bats to humans cannot be ruled out. When spillovers occur from horses to humans the variation observed in HeV variants is minimal.

Identifying Hendra Virus Diversity in Pteropid Bats. (2011) PLoS ONE 6(9): e25275. doi:10.1371/journal.pone.0025275
Hendra virus (HeV) causes a zoonotic disease with high mortality that is transmitted to humans from bats of the genus Pteropus (flying foxes) via an intermediary equine host. Factors promoting spillover from bats to horses are uncertain at this time, but plausibly encompass host and/or agent and/or environmental factors. There is a lack of HeV sequence information derived from the natural bat host, as previously sequences have only been obtained from horses or humans following spillover events. In order to obtain an insight into possible variants of HeV circulating in flying foxes, collection of urine was undertaken in multiple flying fox roosts in Queensland, Australia. HeV was found to be geographically widespread in flying foxes with a number of HeV variants circulating at the one time at multiple locations, while at times the same variant was found circulating at disparate locations. Sequence diversity within variants allowed differentiation on the basis of nucleotide changes, and hypervariable regions in the genome were identified that could be used to differentiate circulating variants. Further, during the study, HeV was isolated from the urine of flying foxes on four occasions from three different locations. The data indicates that spillover events do not correlate with particular HeV isolates, suggesting that host and/or environmental factors are the primary determinants of bat-horse spillover. Thus future spillover events are likely to occur, and there is an on-going need for effective risk management strategies for both human and animal health.

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