Bringing order to the Virosphere

Putative tree of life including viral sequences. The 'sea' of viruses. http://blogs.helsinki.fi/bamford-group/files/2008/06/vevo.png

Physically and evolutionarily speaking we are bathed in a sea of viruses and virus-like elements. Over the last billion or so years these genetic parasites have affected the lives of all kinds of organisms worldwide with sometimes devastating consequences (and others somewhat more positive). An extremely conservative estimate of virus numbers is more likely in the billions and so a major issue is just exactly how can we make sense of all this diversity out there in the Virosphere? 

Determining how one virus – or group of viruses – is related to any other is pretty straightforward nowadays; with sequencing technology and bioinformatic analysis commonplace, we can effortlessly compare nucleotide sequences and generate phylogenetic trees displaying how two or more relate to each other.  This works pretty well in a modern setting when we can compare viral isolates from patients infected in a certain outbreak and determine the path of spread for example over days, weeks, months or years,

Phylogenetic tree tattoo. http://blogs.discovermagazine.com/loom/files/2008/07/origin.jpg

This kind of analysis may however have its limits when we attempt to apply it to virus groups that have potentially diverged millions of years ago – a scenario which would be expected given that viruses have been around with us since the beginning of life itself. Simply over that length of time, any evolutionary ‘signal’ found within a particular gene sequence will have disappeared. As masters of genetic variation, virus genomes can get pretty messy, evolutionary speaking due to horizontal gene transfer, recombination and high mutation rates. How can we then assess the deeper evolutionary questions with regards these highly variable agents?

Krupovic and Bamford (who both specialise in the origins and early evolution of viruses), commenting in the Journal of Virology recently, suggest that we should be focussing our analysis at a different aspect of viral lifestyles: the structural biology of their virions – a feature which they believe reflects the true nature of what a virus really is. This, they say should be suggestive of the early and highly conserved evolutionary relationships (nucleotide sequence may be variable but protein sequence/structure will be more highly conserved). By adopting this strategy, the problems of horizontal gene transfer may also be ablated making all variation observed relative to this gene/protein architecture. Using this perspective, we could infer higher taxonomic structures for the Virosphere reflecting true, ancient biological relationships – that seemingly unrelated viruses infecting divergent host species share a common origin (which has been seen for some human and bacterial viruses).

Picornavirus virion structure made up of viral capsid proteins.  Illustration by David S. Goodsell of The Scripps Research Institute (see this site) [Public domain], via Wikimedia Commons

By attempting to group distantly related viruses, maybe those infecting bacteria, archaea and eukaryotes into common taxonomic classes we should be able to bring more order and structure to the viral universe. – facilitating a better understanding of the entire tree of life. We will not, however be able to accomplish this unless we realise the true age of these viruses and adapt our evolutionary investigations for this. Caveats aside, the idea presented by Krupovic and Bamford should stimulate further evolutionary work to be carried out on more virus taxa.

Order to the Viral Universe. Krupovic and Bamford J. Virol..2010; 84: 12476-12479

Epithelial defence – the role of highly reactive oxygen species

Cell-cell communication is a key feature of biological systems particularly multicellular eukaryotes whose development and functioning rely on complex information exchange between and within cells and tissues and across both long and short distances. The relative roles of particular cell-cell signalling pathways is therefore of interest to a number of processes - one being the host responses during infection.

Fig.1. Typical epithelial cell layer (lung tissue in this case). Nuclei stained in blue, actin in red and tight junctions in green. Notice the physical barrier formation. http://www.seas.upenn.edu/~injury/images/091201%20triple%20slide%20test%20A5%201_5para%2040x%20overlay.jpg

Stopping pathogens at the portal of entry: mucous membrane defence

Fig 1. Mucosal epithelium found within the respiratory tract http://farm4.static.flickr.com/3346/3661529896_fd19543c7f.jpg

Enhancing Oral Vaccine Potency by Targeting Intestinal M Cells.
Azizi A, Kumar A, Diaz-Mitoma F, Mestecky J (2010)

The immune system in the gastrointestinal tract plays a crucial role in the control of infection, as it constitutes the first line of defense against mucosal pathogens. The attractive features of oral immunization have led to the exploration of a variety of oral delivery systems. However, none of these oral delivery systems have been applied to existing commercial vaccines. To overcome this, a new generation of oral vaccine delivery systems that target antigens to gut-associated lymphoid tissue is required. One promising approach is to exploit the potential of microfold (M) cells by mimicking the entry of pathogens into these cells. Targeting specific receptors on the apical surface of M cells might enhance the entry of antigens, initiating the immune response and consequently leading to protection against mucosal pathogens. In this article, we briefly review the challenges associated with current oral vaccine delivery systems and discuss strategies that might potentially target mouse and human intestinal M cells.

Different parts of our bodies are not equally vulnerable to infection and we can consider our immune system to be differentially active across all anatomical sites. One of the biggest sites of pathogen entry is the mucosal epithelium which lines our internal parts including the respiratory, gastrointestinal and urogenital tracts (fig 1.).

These surfaces have been estimated to cover 200x the area that our skin covers and if you think about it, most pathogens will use one of these openings to the outside world to gain entry into our bodies: measles virus via the respiratory tract; HIV through the urogenital tract; and E.coli from the gastrointestinal tract. It is this reason that we expend so much energy and effort in keeping these areas protected from would-be dangerous micro-organisms.

Fig. 2. Dipiction of the mucosal immune system:inductive and effector sites both present with mechanisms of inductive and nuetralisation (SIgA) shown. Notice the MALT complex (M cells, antigen presenting cells and B/T cells). http://www.nature.com/mi/journal/v1/n1/fig_tab/mi20079f1.html

Mucosal immunity is a complex system of anatomical, cellular and molecular 'innate' components: epithelial cells which line the mucosa form very tight layers which prevent bacteria or viruses from penetrating deeper into the body; these cells also secrete a number of antimicrobial compounds and enzymes and can rapidly respond to infection through protein mediated cell-cell signalling establishing intra-cellular protection.

In order to mount an efficient response there is a close association between the mucosal epithelium and lymphoid tissues. This 'mucosa-associated lymphoid tissue' or MALT (examples including nasal-associated lymphoid tissue or NALT and gut-associated ymphoid tissue or GALT) is formed by a close interaction between the epithelial lining (antigen sampling 'M' cells) and underlying immune cell complexes consisting of antigen-presenting cells, T cells and B cells.

These mucosal sites – interestingly – form a collective adaptive unit across the whole body when we consider anti-microbial immunity. This 'common mucosal immune system' is mediated by a network of inductive and effecter sites located within the MALT: inductive sites sensing pathogen activity at one area can generate cellular immune responses which can travel systemically from one site to another (e.g. NALT to GALT) . One of the major players in this immunity is a special type of antibody known as secretory immunoglobulin A (SIgA) which is transported from the underlying immune tissue at effector sites to the surface of the mucosa above and can interact with and bind to pathogens and prevent infection and/or damage being done (see fig2 and fig3).

Interest has stemmed from the thought that if we were able to generate adequate antibody responses at the pathogen site of entry (SIgA at the mucosa) we may able to prevent infection from occurring before it is too late and infection has spread from primary areas to other tissues causing significant disease . Current vaccination strategies have focussed on the development of systemic IgG antibodies without consideration for those found at initial portals of entry and although many have been successful for some diseases, the millions of deaths stemming from pathogen-mucosa related infections warrant further investigation and targeting. The question is which inductive MALT site is best suited for vaccination which in turn depends upon pathogen entry strategy, ease of administration, particular side effects and the relative ability of particular MALTs to develop effecter functions at different mucosal epithelium. To date the most promising of strategies lies with nasal or oral vaccine delivery systems and much research has been carried out in order to improve the efficacy of each.

Fig 3. Cartoon dipicting the general structure of the five basic antibodies in humans: IgA the one concerned with mucosal immunity. http://www.cartage.org.lb/en/themes/sciences/lifescience/generalbiology/physiology/LymphaticSystem/Antibodymediated/AntiBtypes.gif

Novel approaches have been used to target vaccines to those cells responsible for antigenic sampling found across most mucosal surfaces – the M cells. These cells can transport antigenic materials from the epithelial lining to the underlying lymphoid complexes via antigen-presenting cells. These cells however are present in low numbers (1 in 10 million epithelial cells are M cells in the GI tract) with the mucosa hence specific targeting may facilitate higher antigen uptake and greater immune induction and these strategies can be adapted to both respiratory and oral delivery systems depending on the vaccine.

We are only now beginning to see the outcome of current research into mucosal immunity and vaccines. Disrupting the close relationship between pathogens and the mucosa is paramount in preventing the millions of deaths worldwide from mucosal-borne infections and the concurrent development of novel targeting and vaccination strategies we may well witness the generation of highly effective, easily administered and most importantly safe vaccines for the most dangerous known diseases: AIDS; cholera; and influenza.

Azizi A, Kumar A, Diaz-Mitoma F, Mestecky J (2010) Enhancing Oral Vaccine Potency by Targeting Intestinal M Cells. PLoS Pathog 6(11): e1001147. doi:10.1371/journal.ppat.100114

Pathogenesis and cell-cell spread of a deadly virus

Fig.1 NiV infection in an epithelial cell monolayer and different time points and stained (red). Epithelial adhesion indicated in green.

Nipah virus (NiV), an emerging and deadly paramyxovirus of bat origin has been responsible for a number of fatal outbreaks of disease in humans and other animals in South-East Asia and as of yet no vaccine or therapy exists. The mucosal epithelium of the host respiratory tract is believed to be the most common route of viral spread in humans and from this primary site the virus can disseminate systemically through the blood system resulting in rapid endothelial dysfunction and vasculitis. Viruses must overcome a number of cellular mechanical barriers in order to establish efficient infection and knowledge of how they do this is key to understanding pathogenesis and infection in general.

The ability to move between and within cells is central to viral transmission and pathogenesis. Viruses - as obligate intracellular parasites – need to effectively enter and exit target cells within the host organism in which they reside and establish productive infections to facilitate transmission and replication. Viruses generally use two methods of spreading – cell free and cell associated transmission; the first requiring assembly and budding of mature virus from the cell membranes and the second taking advantage of the close association between host cells, facilitating the union of the membranes of two separate cells. The relative roles of each of these processes in vivo and in transmission and infection are not fully known.

The NiV F and G glycoproteins are responsible for virion-host cell membrane fusion and also cell-cell fusion via interactions with NiV receptors expressed in the plasma membrane of target cells. The mechanism of viral spread within the host are thus very important in elucidating how the virus causes disease and transmits itself within a population; polarised epithelial surfaces – where the apical and basolateral domains are structurally and functionally distinct–playing dual roles in initial entry and host-host viral spread.

Fig.2. NiV virion - notice the fusion and attachement proteins on the surface (red and green). http://www.bepast.org/docs/photos/Nipah%20Virus/nipah%20virus.jpg.

Viruses have taken advantage of the fact that host cells require discrete functions on their apical and basolateral domains and can preferentially target mature virus formation to particular cellular locations through intrinsic protein signals built in to their glycoproteins. Apical release will generally facilitate free virion spread and host-host transmission (e.g in the respiratory tract) while basolateral release may allow direct cell-cell spread and could lead to systemic infections not restricted to the particular primary site of replication. NiV can therefore control its transmission within and between hosts by targeting its infection machinery (F and G glycoproteins) to either the apical or basolateral surfaces of polarised cells.

Fig.3. Polarised epithelial cell monolayer. Apical and basal domains shown. http://www-dsv.cea.fr/var/plain/storage/original/media/File/IBITECS/SB2SM/Eq%20Verbavarz/Cell-base.JPG

A recent paper has investigated the mechanism of NiV spread in polarised epithelium in vitro using cell imaging and molecular analyses of its F and G glycoproteins. They demonstrate that the NiV glycoproteins can facilitate cell-cell spread within polarised epithelial cells through targeting to basolateral cell surfaces between closely positioned cells allowing entry to epithelial cells or underlying tissues. They also note that they are also found on the apical domain allowing efficient spread via free virus release. The group explored the functional importance of such targeting by determining the exact molecular signals required for such protein trafficking and then disrupting them. By purposely retargeting the fusion machinery to the apical side, NiV cell-cell spread by lost. The group also report their findings of NiV infection within infected endothelial cells – a major target of NiV – and establish that this interaction may allow viral entry into the central nervous system (CNS).

Their results add to the ongoing the investigation of not only NiV pathogenesis but also to that of other important and deadly viral pathogens. The initial site of viral entry and its subsequent release and spread within a host are essential to the degree of infection and pathogenesis caused by a particular virus in its host. Spread of NiV into sub-epithelial tissues facilitates systemic infection through the host vasculature and leads to the establishment of unique sites of replication in organs systems and tissues far away from the initial portal of entry. Apical dissemination of infectious virus will also allow for host-host transmission via respiratory or urogenital epithelia.

Knowledge of how the virus achieves this spread may allow us to design better vaccines, identify novel therapeutic targets and add to the wealth of information in viral pathogenesis.

Carolin Weise, Stephanie Erbar, Boris Lamp, Carola Vogt, Sandra Diederich, and Andrea Maisner. Tyrosine Residues in the Cytoplasmic Domains Affect Sorting and Fusion Activity of the Nipah Virus Glycoproteins in Polarized Epithelial Cells. Journal of Virology, August 2010, p. 7634-7641, Vol. 84, No. 15

Viral genomes in High Definition

As seen with cellular organisms, our ability to rapidly and accurately sequence multiple viral genomes at low cost is being overtaken by our inability to analyse the functional roles that certain genes – including regions within genes- play in a range of phenotypic processes during an infection. Lack of functional studies represents a major roadblock to understanding the basic biology of these pathogenic organisms and prevents us from generating safe and efficacious vaccines or virotherapies.

High-Resolution Functional Mapping of the Venezuelan Equine Encephalitis Virus Genome by Insertional Mutagenesis and Massively Parallel Sequencing

Brett F. Beitzel, Russell R. Bakken, Jeffrey M. Smith, Connie S. Schmaljohn^*

The United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland, United States of America

We have developed a high-resolution genomic mapping technique that combines transposon-mediated insertional mutagenesis with either capillary electrophoresis or massively parallel sequencing to identify functionally important regions of the Venezuelan equine encephalitis virus (VEEV) genome. We initially used a capillary electrophoresis method to gain insight into the role of the VEEV nonstructural protein 3 (nsP3) in viral replication. We identified several regions in nsP3 that are intolerant to small (15 bp) insertions, and thus are presumably functionally important. We also identified nine separate regions in nsP3 that will tolerate small insertions at low temperatures (30°C), but not at higher temperatures (37°C, and 40°C). Because we found this method to be extremely effective at identifying temperature sensitive (ts) mutations, but limited by capillary electrophoresis capacity, we replaced the capillary electrophoresis with massively parallel sequencing and used the improved method to generate a functional map of the entire VEEV genome. We identified several hundred potential ts mutations throughout the genome and we validated several of the mutations in nsP2, nsP3, E3, E2, E1 and capsid using single-cycle growth curve experiments with virus generated through reverse genetics. We further demonstrated that two of the nsP3 ts mutants were attenuated for virulence in mice but could elicit protective immunity against challenge with wild-type VEEV. The recombinant ts mutants will be valuable tools for further studies of VEEV replication and virulence. Moreover, the method that we developed is applicable for generating such tools for any virus with a robust reverse genetics system.

Beitzel et al recently publish the development of a ‘high-resolution genomic mapping technique’ that will facilitate the easier investigation of particular viral genes in a range of virus/host interactions. Venezuelan equine encephalitis virus, endemic to South America, is an important zoonotic pathogen that continually re-emerges from its rodent reservoir via a mosquito vector causing potentially fatal encephalitis in both humans and horses. There is currently no vaccines or anti-viral treatments for this disease and the exact roles that each of its 8 proteins play in its basic biology and replication are currently being investigated, hoping to yield helpful insight.

Studying the role of nsP3 gene in VEEV but later expanding it the rest of the genome, the group used a technique called transposon mutagenesis in which a short (15 nucleotides) DNA fragment is inserted randomly into the target gene or genome being studied. They generated a large cDNA library with high coverage in that every nucleotide had been targeted around 200 times and using high- throughput DNA sequencing were able to map the inserts at a high resolution and record the frequency that they were present at. Combining this with a reverse genetics system, they generated recombinant infectious virus carrying and expressing the inserts in order to assess their function in replication and initially, the group assessed the role that these inserts had on replication at particular temperatures.

By altering the temperatures in which the viruses were grown they were to able to effectively screen for a particular phenotype (the ability to grow at 40 degrees for example) and easily map the locations that would and would-not tolerate the 15bp insertions. Using this method they generated temperature sensitive (ts) mutants that were able to replicate at one temperature (30) and not at another (40).