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Attempting to understand biology through the study of viruses, how they spread, cause disease and how we can apply our knowledge. With love from Belfast, Northern Ireland
I've talked about this before (here with mumps virus) but just what makes vaccines so good at being vaccines? - that is, what makes them safe yet immunogenic? The great thing about this is that maybe if we understand these processes a bit better we may even be able to develop safer, more effective and cheaper vaccines that will ultimately save more and more peoples lives. A lot of research at the minute is cuurently attempting to tease apart the roles of hundreds of virus genes in infection and disease with many groups focusing on specifically how these genes affect attenuation, that is how they make the virus weaker and less dangerous so it can be used as a vaccine.
So it starts off like this: we know vaccines are safe and effective when after we give them to people/other animals very little get sick and a lot of them are immunized against infection with that particular virus yet the 'wild-type', normal virus is able to infect and cause disease - so just what makes these different? Well to find out we have to compare the two viruses - the weak one and the virulent one and somewhere in their genomes lie differences that will possibly shed light on the important molecular mechanisms behind virus attenuation and pathogenesis.
Canine distemper in dogs. http://www.dogs-info.net
In a recent paper, Dietzel et al investigated these processes using a great model system, specifically canine distemper virus - or CDV, a virus able to infect and cause life-threatening disease in a whole range of mammalian species in a domestic or wild-life situation and hence is obligatorily given as part of a multivalent vaccine to all domestic dogs. The group began by comparing the matrix - or M- gene between a vaccine strain of a virus known as canine distemper virus or CDV and a wild-type one. This M gene encodes a protein which plays a major role in the assembly of new virus particles, cell-cell spread and particle stability within CDV-infected cells through co-ordinating genome interactions with virion-surface proteins. Its role in CDV pathogenesis - and other viruses - has barely been looked at. The two M proteins differed at only six different amino acid positions (3%) so to look into the role of them both during CDV infection the group genetically engineered a CDV virus based on the wild-type but carrying the vaccine-derived M gene in place of its own. The growth and physical characteristics of the three viruses :wild-type, wild-type + vaccine M and vaccine strain viruses were looked at as well as physically characteristics of the produced particles, how they are assembled and what happens during animal infection.
The 3 viruses grow equally well in cell culture conditions.
Despite the three viruses growing equally well under cell culture conditions (see growth above and how the infection looks in tissue culture - all are pretty much similiar), the two differed markedly in their particle-infectivity ratio, that is - the number of virus particles capable to carry out a successful infection compared to the total number of particles in a given volume, i.e some particles may be non-functional for some reason, maybe because they are less physically stable or damaged.
virus particle release from the tops or bottoms of the cells
The vaccine virus also differed in the way new CDV particles were released from cells: the wild-type virus is released from the tops of polarised epithelial cells while the vaccine strain is released from both the top and the bottom. However, transfer of the vaccine M gene wasn't able to confer bipolar release onto the wild-type virus indicating the function of other CDV genes in this phenotype. To determine a mechanism for this bipolar release the group analysed the intracellular distribution of the key CDV proteins, H and F involved in particle production and cell-cell spread. They found that this proteins differed in their location between the three viruses: the wild-type only had them on the tops of cells while the vaccine strain had them on both sides, the chimeric virus had an intermediate distribution. These results highlight the primary ability of the CDV matrix protein to coordinate protein distribution within the cell.
CDV protein intracellular distibution
All these results are good and all but what effect do they actually have in vivo - in a situation where the virus is infecting multiple cell types and is constantly battling the immune system? To determine the effect of vaccine M gene, groups of ferrets (an excellent model of CDV infection that are naturally infected by the virus) were inoculated intranasally with the three viruses and disease severity, body temperature, disease symptoms and fatality was recorded. Interestingly, based on these measures, the wild-type with the vaccine M was more attenuated that the vaccine virus as shown below with the measure of ferret blood leukocytes - the more blood cells the less pathogenic the virus.
A measure of CDV disease - the percent of ferret blood cells - a CDV target cell
The process of pathogen attenuation for the generation of protective vaccines has worked well in the past for many diseases yet many viruses and bacteria do not prove to be responsive to this method. Other more targeted processes may have to be designed in order to create vaccines for against these diseases. Work investigating the molecular basis of virus pathogenesis and attenuation may allow us to rapidly and successfully attenuate these hard-to-vaccinate viruses in a more targeted fashion - and one that may work better than the previously tried methods.
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Dietzel, E., Anderson, D., Castan, A., von Messling, V., & Maisner, A. (2011) Canine Distemper Virus Matrix Protein Influences Particle Infectivity, Particle Composition, and Envelope Distribution in Polarized Epithelial Cells and Modulates Virulence. Journal of Virology, 85(14), 7162-7168. DOI: 10.1128/JVI.00051-11
One method that has been used extensively to generate worthwhile vaccines is that of forcing an initially disease-causing virus to replicate inside a non-natural cell, for example imagine forcing the human specific measles virus to replicate within cells from a chicken. Over time these viruses - all with extremely high mutation rates - will evolve and adapt to the conditions within a chicken cell while at the same time losing it's ability to survive within human cells. The use of these live-attenuated viruses as vaccines has led to the dramatic reduction in a number of important human - and livestock - viruses - the likes of measles, mumps, polio and rinderpest.
These vaccine viruses usually retain their ability to infect and replicate within their host (one of the reasons why they are so good at protecting us) yet fail to cause significant disease past the odd fever. However, in some cases the use of these vaccines has led to a number of cases where they caused serious illness. Hence, with the powerful immunity these vaccines generate comes the important potential chance of causing disease and therefore the ability to understand and predict how a particular vaccine will behave following administration is key to continuing the safe use of live attenuated vaccines. Sadly, our knowledge of the mechanisms behind virulence and attenuation are largely unknown.
Vaccination of the man behind the mumps vaccine - Maurice Hilleman's daughter, Kirsten, while her half-sister, Jeryl Lynn, looks on. The MuV vaccine strain Jeryl-Lynn was originally developed from the virus which infected Hilleman's daughter of the same name through non-natural replication. http://www.historyofvaccines.org/
One example of this can be found with the case of the mumps virus (MuV) vaccine. Interested in mumps? see here. Before the widespread use of the vaccine, MuV was responsible for the majority of cases of aseptic meningitis (inflammation of the lining of the brain not caused by bacteria) in the western world - this virus is highly adapted in entering the human central nervous system and replicating within the epithelial cells that line its inner layer, making it particularly dangerous. Because of this, any vaccine batch produced must go through rigorous pre-clinical testing, the majority of which is carried out in primates. Yet sometimes, vaccine viruses will lead to the development of aseptic meningitis following immunization. We still do not adequately understand the molecular basis of the ability of MuV to cause disease in the nervous system - something that may facilitate the development of new and improved MuV vaccines.
Sauder et al, publishing in Journal of Virology, explore the genetic basis of MuV neuropathogenesis through the generation of around 30 chimeric viruses - see below - comprising genes of a neurovirulent MuV (strain: 88-1961) and a highly attenuated MuV (vaccine strain Jeryl-Lynn 5). Through the analysis of the potential for these viruses to cause disease in a rat model of mumps meningitis they were able to assess the contribution of specific genes - or combinations of genes - in either increasing or decreasing its ability to cause disease.
Chimeric mumps viruses (combinations of attenuated - JL and virulent - 88). What effect will each have on pathogenesis?
MuV has a single-stranded RNA genome of 15,384 nucleotides and encoded within this one molecule are 7 genes through which at least 9 different proteins are expressed. These proteins - and hence their corresponding genes - govern the basic biology of this virus: building of the virus particle, receptor binding, cell entry, transcription and replication and finally, cell exit. N, P and L forming the replication apparatus of the virus while M, F, SH and HN are involved in particle assembly, entry and exit. It is these same proteins that are responsible for the ability of MuV to cause disease in humans, specifically aseptic meningitis. Any understanding of the ability of a virus to cause disease must identify its key molecular - and genetic - components hence, what particular genes along the MuV genome are responsible for causing aseptic meningitis in humans? Is it those that allow the virus to enter the cell? Those involved in replication? or those involved in building the virus particle? Sauder et al sought to try and convert an attenuated virus into a virulent one and vice versa - and in doing so uncover the biology behind MuV pathogenesis.
Can we transform the virulent 88 strain to an attenuated virus by inserting combinations of attenuated JL genes?
Above shows the results from the initial experiment comparing disease caused by the different viruses: This is where the added different genes from the attenuated virus to the virulent to see whether or not this weakened the viruses ability to cause disease. As you can see, no individual genes or combinations added to the 88-1961 virus caused it to be as attenuated as the vaccine strain, suggesting that in this case with MuV attenuation is a complex, polygenic trait involving many genes. Neither the transfer of all replication proteins (N, P and L) nor the assembly proteins (M, F, SH and HN) resulted in complete attenuation. The most dramatic effect was seen with N and M transfers - although the reason why was not addressed.
Can we transform the attenuated JL strain to a virulent virus by inserting combinations of pathogenic 88 genes.
Following on from this, the group tried to see whether they could turn the attenuated into the virulent virus through carrying out the reverse of the above experiment although this time the results were not the same as no genes or combinations resulted in anywhere near the levels of pathogenesis seen for the 88-1961 virus. Even with the addition of the previously effective N and M combination.
These results tell us a number of things about MuV attenuation and virulence, firstly: it's a lot more complex than we might have previously thought! - this is not all down to one gene but a few of them working together in combination. Secondly, it doesn't work both ways - the mechanisms behind how a virus causes and doesn't cause a disease are different as the same genes couldnt do both. And lastly these results point us to some interesting avenues of future research into virus attenuation - what are the specific molecular roles each of these genes play in turning the virulent virus into an attenuated one? For example, why do the attenuated N and M have such a drastic effect? More work will be undoub... Read more »
Sauder CJ, Zhang CX, Ngo L, Werner K, Lemon K, Duprex WP, Malik T, Carbone K, & Rubin SA. (2011) Gene-specific contributions to mumps virus neurovirulence and neuroattenuation. Journal of virology, 85(14), 7059-69. PMID: 21543475
Everyone is aware of the ability of our immune system to defend against microbial pathogens yet its role in the prevention of other diseases - like cancer - is generally over-looked. Yet it is through the harnessing of our immune system that novel ways of combating cancer may arise. And interestingly enough, through the use of engineered viruses - the same ones our immune system protects against - we may now control aspects of immunity to suit these medical needs. Kotke et al, report in Nature Medicine just this week, their use of a new virus-based immunotherapy platform that was able to effectively 'cure' mice suffering with cancer.
One of the hallmarks of cancer appears to be the ability to persist in the face of an active immune system - see fig. 1. Newly cancerous cells and tumours are able to survive and proliferate without - or at least protecting themselves - against a full-blown immune attack. Our immune system is usually able to protect us from the development of cancer but in some cases something fails and the result is more often than not - cancer.
Fig 1. Newly recognised cancer hallmarks - note avoiding immune destruction. (Hanahan and Weinberg 2011).
Through the recognition of 'tumour antigens' (proteins expressed only on cancer cells) or 'tumour-associated antigens' (proteins expressed differently on cancer cells), our immune system is usually able to mount an effective response toward those cells. This is the system that tumours are able to suppress yet we may be able to boost the natural immunological nature of tumours in order to cure them. The discovery of these proteins - just like those found on the surface of virus particles or bacterial cells - may allow us to effectively vaccinate people against cancer, allowing their own immune system to remove the cancerous cells.
This is what we try and achieve through cancer vaccines and immunotherapies. Kotke et al set about trying to improve upon these current immunotherapy platforms, which - as they state - suffered from a number of problems including: lack of known tumour antigens and coverage of only a few such proteins. Most current immunotherapies rely upon the immunization with only a single antigen. Previous work by the group showed that if you kill normal cells from a patient in vivo - you may be able to elicit an effective anti-tumour immune response through the induction of tumour-associated antigen immunity (when cells die they either burst and release their insides).This work effectively showed that you could immunize with a wide range of tumour-associated antigens from normal cells and protect against cancer - both circumventing the above two problems.
VSV particles - www.standford.edu
To improve upon this model, they developed a virus-based platform for the expression of a wide range of tumour-associated antigens in vivo termed altered self antigen and epitope library (ASEL) - see fig 3. They based their method upon the vesicular stromatitis virus - or VSV - normally a virus solely of livestock that also has the ability to infect humans. In humans it causes a generally mild flu-like illness and may form vesicles on the skin. Although a single-stranded non-segmented negative sense RNA virus, we have the ability to generate infectious VSV particles entirely from cDNA plasmids encoding the entire VSV genome. This has facilitated the development of VSV as a key eukaryotic expression vector for multiple uses, such as these cancer immunotherapies and specifically, as an oncolytic treatment. Using standard molecular biology techniques (PCR, restriction enzyme digests and ligations) you can insert any gene from whatever source you want into the VSV genome and it will be expressed inside cells following infection. The benefit with using this virus is that even without the expression of tumour antigens from it's genome, replication within a cell will kill the cell anyway. It is a double hit strategy.
Fig.3. Cloning the cDNA library into the VSV genome in forward and reverse orientations = VSV-ASEL library
In order to express hundreds of tumour-associated antigen genes, the group used reverse-transcriptase PCR to amplify all expressed genes from normal prostate tissue and inserted the entire normal prostate tissue cDNA library into VSV. They were then able to infect mice that suffered with prostate cancer and observe what happened to their tumours - specifcally, was an effective immune response generated and did the tumour shrink? VSV virus particles were injected into the mice, virus entered the cells of the mice and began to replicate and express their genes, including the newly inserted prostate cDNA. Essentially, thousands of virus particles were adminsired, each containing a slightly different gene from the prostate cDNA library. High levels of tumour associated antigens were therfore being expressed in mice allowing for the generation of an effective immune response.
Survival of mice treated with the VSV viruses - GFP expressing negative control; and the VSV ASEL in mice with established 'TC2' prostate tumours.
This approached effectively cured the mice who suffered from prostate cancer. Following this treatment a number of resistant tumours emerged which were again subjected to a further treatment using a cDNA library taken from the tumour itself this time and this readily treated the secondary resistant cancers. The clinical benefits of this approach can hardly go unnoticed. The ability to administer a broad tailored therapy that has the potential to cure an established tumour will be revolutionary, especially given the relative ease at which this can be developed 'at the bedside'. The ability to easily genetically manipulate viruses has - and will continue to - revolutionise the medical sciences. Look out for the eminent clinical trials.
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Kottke, T., Errington, F., Pulido, J., Galivo, F., Thompson, J., Wongthida, P., Diaz, R., Chong, H., Ilett, E., Chester, J.... (2011) Broad antigenic coverage induced by vaccination with virus-based cDNA libraries cures established tumors. Nature Medicine. DOI: 10.1038/nm.2390
During the last century, mass vaccination campaigns were rolled out across much of the industrialised world. And, with much success, we have near eradicated most common virus infections from these populations. Despite the more recent efforts to bring the developing world into this fold, major setbacks have been uncovered, one being: how exactly are we to stably transport delicate vaccine stocks from their place of production to the regions that need it most.
Many of these vaccines, known as 'live attenuated vaccines' - or LAVs-are generally unstable in the environment outside of our cells and bodies. For example, the measles virus has an outer lipid membrane that is highly delicate and damage to this removes any chance of this vaccine working. Basically, these LAVs, which rely upon a weaker infection, cannot successfully complete their replication cycle without an envelope. Generally, the ability of a vaccine to provide protection is intrinsically linked to its overall structure; you destroy the structure, you destroy protection.
Unstable measles virus particles
The more difficult places to transport vaccines to happen to be more tropical regions, like sub-Saharan Africa, South America and South-East Asia. These places are very warm and humid which goes against what our vaccines like. With temperatures up to 40 degrees Celsius and the fact that freezing also destroys the vaccine, the only way forward is the implementation of a cold chain. If we keep the vaccine stock 'chilled' from manufacture (see Merck or the Serum Institute of India) all the way to administration, then we have a chance to prevent its degradation. But this is not as easy as it may sound as it is economically and logistically difficult to achieve this in developing countries. To overcome this, the produced vaccine stock is freeze-dried to remove any water that may contribute to its instability and when this prep reaches the clinic it is then reconstituted through the addition of a solvent preparation, which has then all got to be kept cool.
With measles virus vaccines however, even upon reconstitution, there are still major losses to immunogenicity and this problem is compounded with the use of multi-dose vials of vaccines. A single vaccine stock, prepared in the morning may now sit in the clinic unrefrigerated until the end of clinic hours. If we were then to get immunized at 5:00 pm, what are the chances of that being successful? It is thus not much of a surprise to hear that many virus outbreaks have been attributed to breaks in the cold chain like this. Our ability to eradicate many diseases in these countries and hence worldwide, is blocked by difficulties in the cold chain.
Being so, Bill and Melinda Gates identified "Vaccines That Do Not Require Refrigeration" as one of their funded 14 Grand Challenges in Global Health and the results of one potential solution to this problem have just been published in the journal Vaccine (read it here). This work aimed at identifying novel reconstitution liquids that would increase the thermostability of the prepared vaccine and therefore increase its 'shelf-life' in these more difficult environments. Using a recombinant measles vaccine virus that expresses green fluorescent protein (GFP) upon infection, the group were able to assess the level of infectivity of a range of vaccine stocks reconstituted in varied solutions.
Counting measles virus infected (GFP positive) cells
Indeed, they tested >11,000 formulations (myriad combinations of buffers, stabilizers, solubilizers, preservatives, pH and tonicifiers - whatever they are?) using their recently developed high-throughput system in which they were able to automate the whole process. These results were validated and confirmed with another virus, this time an adenovirus expressing GFP; results were the same. The group identified a formulation that caused the vaccine to "suffer <1.0 log loss after 8 h at 40 ◦ C in the liquid state", a major improvement on a previous loss of" 1 log of potency after 8 h at 37 ◦ C in the reconstituted (liquid) form". In conclusion, they identified a novel vaccine formulation that substantially increased the stability of two viruses, which can be used in immunization campaigns worldwide. As I've alluded to before, the use of high-throughput screens has much potential in the world of virus research.
Log loss of infectivity of vaccines
How is this result likely to change vaccine implementation in the real world then? Well, the evidence presented here indicates the ability of creating more stable vaccines that would transfer to better vaccine coverage yet the real problem appears to be whether current vaccine manufacturers would accommodate such a significant change to production, especially given their inherent unwillingness to invest in the developing world.
The cost associated with this may be offset by the savings made through reduction in vaccine wastage and loss of cold chain implementation but we must realise that only when such an improvement looks attractive to the market will change come. The future of vaccination looks to be bright, especially with the combination of newer technologies and the backing of large philanthropic organisations.
... Read more »
Schlehuber LD, McFadyen IJ, Shu Y, Carignan J, Duprex WP, Forsyth WR, Ho JH, Kitsos CM, Lee GY, Levinson DA.... (2011) Towards ambient temperature-stable vaccines: The identification of thermally stabilizing liquid formulations for measles virus using an innovative high-throughput infectivity assay. Vaccine. PMID: 21616113
Two papers published recently explore the idea of targeting antiviral compounds to the host cell using high-throughput organic synthesis and an in vitro screen. Both groups identify a single novel inhibitor of virus replication and both are host specific. One paper goes further and identifies the target and also the in vivo potential of this compound. These papers highlight the hopes and pitfalls of targeting the host cell against viruses while shedding light on the basic biology of these viruses.
We can protect ourselves from the harmful effects of viral infection through the use of vaccination, antivirals or indeed behavioral changes. Yet not all viruses have proved to be amenable to these and some, like measles and mumps, are quite easily combated with mass immunization campaigns while the likes of HIV have shown to be quite sensitive to antiviral compounds.
Respiratory infections are a problem! hastings.gov.uk
For a majority of viruses though, we have neither effective vaccines nor antivirals. We need these now more than ever. And, even those which we do have vaccines and/or antivirals (Influenza A), resistance is quickly built up over time rendering them clinically useless. How then are we able to generate new, more effective antiviral molecules against a whole range of viral pathogens, especially given the cost of taking a single drug to patients?; we want more bang for our buck with antivirals. One way maybe through the targeting not of a viral gene or protein but a cellular one. The virus life cycle is so intimately connected to that of the host cell that removal of cellular pathways may have a dramatic effect on virus functioning and applications like these have the beauty of possibly targeting both established and emerging pathogens.
Krumm et al, from the Emory University School of Medicine, Atlanta, U.S.A, demonstrate the feasibility of this approach using viruses from the paramyxo- and the orthomyxoviruses (generally called the 'myxoviruses') as their model of choice. Read the paper here. Using this, they discover a novel compound that demonstrates host-cell specificity while greatly inhibiting virus replication. Even at nanomolar concentrations (potent!).
This work falls on the back of a series of experiments they reported back in 2008 where they screened a 137,500 compound library against in vitro infection with the related measles, canine distemper and human parainfluenza 3 viruses. This was to determine if they could uncover broad range, potent inhibitors of a large number of viruses. Moving away from the 'one-bug, one-drug' paradigm, this work identified a class of 11 compounds that did just that: they significantly inhibited replication and cytopathic effect of all the three viruses. They use the data from this series of experiments to find a host-targeted compound.
They state that their 'ideal' inhibitor would be:
In search of candidates with a host-directed antiviral profile, we anticipated three distinct features of desirable compounds: a) potent inhibition of virus replication at the screening concentration (3.3 µM); b) a primary screening score, representative of the selectivity index (CC50/EC50), close to the cut-off value for hit candidates due to some anticipated host-cell interference ( = 1.9); and c) a broadened viral target spectrum in counter screening assays that extends to other pathogens of the myxovirus families
initial compound A) and B), the synthetically optimised JMN3-003
A single compound was taken forward and then synthetically optimized (high-throughput synthetic chemistry anybody?) to achieve even better inhibition. No longer do we have to rely on the natural diversity of compounds out there. This new and improved compound (JMN3-003) could inhibit not just myxo-virus replication but also that of clinically relevant RNA (Sindbis virus) and to a lesser extent DNA viruses, such as vaccinia virus. The broad activity of the molecule suggests that it may target a more general aspect of viral replication than others had previously. The activity of this drug was also host-cell specific, suggesting that it did not target a virus structure but rather one found within the host cell, whose activity would change depending on the species assayed.
JMN3-003 antiviral activity
This molecule had little or no effect on the metabolic activity of the immortalized cell lines used or of other primary cell cultures when administered at >7,000X the active dose, possibly hinting at the safety profile of such a drug. And, under liver cell extract stability tests, the compound showed a desirable extrapolated half-life of around 200 minutes. Host transcription and translation weren't affected by drug administration.To be safe, the authors put forward that this compound may only be preferentially used in acute respiratory infections where the treatment time would be substantially shorter. This group did not carry out any in vivo work so the real safety of this compound cannot really be assessed. See below for how this should be done.
Activity changes depending on the cell used. Human PBMCs or primate Veros
So, we have demonstrated that the compound inhibits virus replication. But just how exactly does it do this? At what stage of the virus replication cycle does it act? Entry? Transcription? replication? assembly? exit? The group addressed these points through the use of assays that measure virus envelope fusion with the cell membrane (virus entry) and virus gene expression and replication (post entry). This work showed that fusion was not inhibited when the drug was added suggesting a post entry step was being targeted. When compared to a known inhibitor of virus polymerase activity, JMN3-003 looked very similar. Other experiments, assessing levels of mRNA and genomes produced from virus replication showed a significant inhibition in one or both processes during measles or influenza virus infection. Some component associated with virus polyme... Read more »
Bonavia, A., Franti, M., Pusateri Keaney, E., Kuhen, K., Seepersaud, M., Radetich, B., Shao, J., Honda, A., Dewhurst, J., Balabanis, K.... (2011) Organic Synthesis Toward Small-Molecule Probes and Drugs Special Feature: Identification of broad-spectrum antiviral compounds and assessment of the druggability of their target for efficacy against respiratory syncytial virus (RSV). Proceedings of the National Academy of Sciences, 108(17), 6739-6744. DOI: 10.1073/pnas.1017142108
Krumm, S., Ndungu, J., Yoon, J., Dochow, M., Sun, A., Natchus, M., Snyder, J., & Plemper, R. (2011) Potent Host-Directed Small-Molecule Inhibitors of Myxovirus RNA-Dependent RNA-Polymerases. PLoS ONE, 6(5). DOI: 10.1371/journal.pone.0020069
Yoon, J., Chawla, D., Paal, T., Ndungu, M., Du, Y., Kurtkaya, S., Sun, A., Snyder, J., & Plemper, R. (2008) High-Throughput Screening--Based Identification of Paramyxovirus Inhibitors. Journal of Biomolecular Screening, 13(7), 591-608. DOI: 10.1177/1087057108321089
The emergence of a deadly Ebola virus (EBOV) strain into human populations has been our constant worry for over 40 years now. And news of the most recent case where a 12-year-old girl was killed near a major trade hub in Uganda earlier this month only serves to remind us of the devastating impact it can have. Infection with the Zaire strain of EBOV, the one that caused the death of the girl, initially causes a rapid onset of fever and headaches culminating in internal and extrernal bleeding, vomiting and diarrhea and eventually, death. No specific therapy or vaccines are available.
While Ebola virus Zaire is known to cause up to 90% mortality the exact mechanisms of how it causes disease in humans are not understood. Kondratowicz et al, publishing recently in the journal PNAS, add to our knowledge of EBOV pathogenesis and biology by identifying the receptor molecule (TIM-1) on the surface of our cells that the virus uses to infect humans.
What is a virus receptor?
EBOV glycoprotein (GP) synthesis and functions. http://www.bio.davidson.edu/
Viruses are parasites of cells; that is, in order to survive they need to enter our cells and replicate. One major barrier to this, however, is the cell's plasma membrane: an outer covering made up of fats and proteins that protects the cell from the harsh outside environment. On the other hand, viruses have evolved diverse strategies to bypass this barrier and EBOV is not any different (reviewed in TWiV here). Ebola glycoproteins (GP) 1 and 2 lie on the outside of the virus particle and mediate both attachment to the cell via a receptor molecule AND fusion of the virus membrane with the cell membrane releasing the infectious virus genome into the host cytoplasm. This process also appears to involve a peculiar process known as macropinocytosis.
Viruses do not infect every cell within the human body - they are pretty selective, having adapted their replication cycle to a particular host over hundreds or thousands of years. What then dictates this selectivity? and what role does this host cell choice have on the replication of the virus? The answer lies upon the particular receptor moleculas the viruses use.
EBOV GP structure. http://www.als.lbl.gov/
What about the ebola receptor?
A number of cell surface molecules have been identified that play a role in the complex process of EBOV entry, all mainly only increasing its efficiency. Although none have been shown to specifically interact and bind to the virus GP 1/2, these may play more of a general role in enhancing entry. How then are we meant to identify what molecules act as specific receptors? More specifically, what does a virus receptor behave like? if we saw one, what would it look like?
To prove that a particular molecule is a receptor for a specific virus, it should fulfil a set of predictions. Primarily, it must:
Physically interact with virus proteins (GP 1/2) on it's surface
Expression of it must significantly enhance infection (shoudn't be found on any cells it can't infect) and if it cannot infect a particular cell, does forced expression restore infectability?
Removal or blocking of receptor molecule will prevent infection of cell previously susceptable
How did the group do this?
To search for the receptor molecule that EBOV uses, Kondratowicz et al sought to correlate EBOV infection with the expression of the receptor molecule. Using a non-related virus (Vesicular Stromatitis virus) that was engineered to express EBOV glycoproteins as well as a green fluorescent protein (GFP), they infected a panel of 54 tumour cell lines. By counting the number of green cells (i.e those that EBOV could enter) they could see which cells were most easily infected and hence expressed the required receptor molecule.
Percent transduction (GFP expression) of the VSV-EBOV-GP virus (A), Non-EBOV-VSV (B) in a range of cell lines. Correlated with TIM-1 expression date (C).
TIM-1 on the cell surface
The group then searched through gene expression data corresponding to the cell lines and attempted to pull out a set of candidate receptors common to all susceptible cells, i.e. those that generated a lot of green cells. One gene that they found to be significantly correlated with infection was TIM-1, a type one transmembrane protein expressed on dividing kidney cells and some immune cells. They do note that TIM-1 is not found on all the susceptible cells, suggesting a role for other unknown factors in EBOV entry. Alas the hunt may continue.
Kondratowicz et al further provides ample evidence that TIM-1 plays a significant role in EBOV glycoprotein mediated entry: he team showed that if you knockdown expression of TIM-1 on the surface of infectable cells with siRNAs, no longer does EBOV infect the cells; they also showed that expressing TIM-1 in cells not previously able to be infected, or ones that weren't all that good at it, allowed the entry of EBOV. Soluble TIM-1 is able to outcompete cell surface TIM-1 for binding to EBOV GP thus causing an inhibition of entry. TIM-1 EBOV GP binding is also demonstrated, fulfilling the requirement of direct interaction between the two. They go on to identify the expression of TIM-1 on the epithelial cells lining the human respiratory tract, a fact important as the means that EBOV initially infects us. Interestingly, the receptor is also found in conjunctiva around the eye. And, finally, infection with viable 'live' EBOV (not the VSV + EBOV glycoproteins) was blocked by administration of an anti-TIM-1 antibody.
We may safely conclude that TIM-1 is at least one of the receptors... Read more »
Kondratowicz AS, Lennemann NJ, Sinn PL, Davey RA, Hunt CL, Moller-Tank S, Meyerholz DK, Rennert P, Mullins RF, Brindley M.... (2011) From the Cover: T-cell immunoglobulin and mucin domain 1 (TIM-1) is a receptor for Zaire Ebolavirus and Lake Victoria Marburgvirus. Proceedings of the National Academy of Sciences of the United States of America, 108(20), 8426-31. PMID: 21536871
The inter-species transmission of viruses and other pathogens (see data below) poses a serious threat to public health, the global economy as well our environment and biodiversity. Just look at Ebola; SARS and Hendra viruses. With the numbers of emerging viruses increasing year on year, how best are we to deal with this incoming threat? As they say, the most effective bioterrorist is nature herself; so how can we stop her?
Increasing numbers of emerging infectious diseases. Jones et al (2008)
First of all, we can identify a number of possible routes that may be followed if we are to successfully combat or at least limit human infection with other animal viruses. These would include:
Identifying what viruses may make (or are in the process of making) the jump into humans, i.e. what viruses are out there?
What are the molecular mechanisms behind inter-species transmission and adaptation?
Can we identify temporal and geographical (or cultural) 'hotspots' that correlate with increased risk of virus emergence?
How can we develop potential vaccines/antivirals to further protect vulnerable local/global populations?
And, perhaps most importantly - and the most difficult aspect:
How are we going to fund this and what are the most cost-effective measures of doing this, i.e can we identify the best places to protect ourselves and place our resources there?
Hantavirus - a deadly group of re-emerging viruses. http://virology-online.com/
Orrock et al, publishing recently in the journal American Naturalist, give their contribution to this complex virus protection scheme by identifying key ecological regulators of the potential emergence of a fatal human virus from its rodent species reservoir. Knowledge of this may allow us to pin-point potential danger areas in terms of countries/regions or seasons (by applying their principles to other viruses and ecosystems), which would increase the risk of human infection. By placing emphasis on these areas we could develop a safer, more cost effective strategy to protect at-risk populations.
The model system the group used was that of the rodent-borne hantvirus, Sin Nombre virus (SNV) infecting its host, the deer mouse, Peromyscus maniculatus on the Californian Channel islands. This virus was only relatively recently found to be present among Channel islands deer mice. Hantaviruses are a group of trisegmented negative sense RNA viruses that naturally infect rodent species around the world. Two groups are recognised: one found throughout the new world and the other, throughout the old. When a human is infected by one of the new world viruses (Sin nombre virus, for example), they may develop what is known as hantavirus cardiopulmonary syndrome (HCPS), a life-threatening disease (with up to 50% mortality) caused by leakage of fluids into the lungs. Humans get infected through coming into contact with infected rodents through their aerosolised urine, feces or saliva. In the U.S, the deer mouse (Peromyscus maniculatus) is the primary reservoir of this viruses. Environmental determinants of rodent density are thought to play a significant role in the risk of rodent-human disease through increasing the chances of human/deer mouse contact.
Specifically, they obtained SNV-specific antibody data relating to infected mice across all islands, giving them an accurate estimate as to the prevalence of SNV in these mouse populations. The group also compiled data corresponding to a number of environmental factors of these islands, including: area, perimeter, elevation, annual precipitation ( a good correlate with island productivity) and finally the number of deer mouse predators found across the islands. These numbers allowed Orrock et al to determine which ecological factor correlated well with SNV prevalence individually or in combination with others; data that would allow for the prediction of at-risk areas across the islands.
A number of factors were identified, for example: SNV prevalence correlated well with annual precipitation on the islands as well as island area, which I guess may be expected given that these factors influence the food sources that the mice eat as well as potential space to leave and breed.
Predator richness negatively correlates with SNV prevalence, suggesting that if we were to artificially remove top predator species from this ecosystem, rodent population density would increase, leading to more and more SNV-infected mice with a greater chance of infecting both themselves and humans. The authors state that the protection of both biodiversity and individual predators within ecosystems would serve to protect human populations from rodent-human virus transmission through the better regulation of host density. Also, environmental increases in primary productivity within the isalnd may also increase the risk of emergence.... Read more »
Orrock, J., Allan, B., & Drost, C. (2011) Biogeographic and Ecological Regulation of Disease: Prevalence of Sin Nombre Virus in Island Mice Is Related to Island Area, Precipitation, and Predator Richness. The American Naturalist, 177(5), 691-697. DOI: 10.1086/659632
The interferon (IFN) signalling pathway acts as a primary defense against all viruses through the induction of expression of hundreds of genes following infection; the exact functions of each are, at best, poorly understood. In order to gain a better insight into the antiviral mechanism of the induced genes, Schoggins, et al. (2011) performed a sensitive high-throughput screen of the effects of each one on infection with a range of RNA viruses.
Structure of the IFN-alpha protein. http://www.wikipedia.com/
The type I interferon response protects cells against invading viral pathogens. The cellular factors that mediate this defence are the products of interferon-stimulated genes (ISGs). Although hundreds of ISGs have been identified since their discovery more than 25 years ago1, 2, 3, only a few have been characterized with respect to antiviral activity. For most ISG products, little is known about their antiviral potential, their target specificity and their mechanisms of action. Using an overexpression screening approach, here we show that different viruses are targeted by unique sets of ISGs. We find that each viral species is susceptible to multiple antiviral genes, which together encompass a range of inhibitory activities. To conduct the screen, more than 380 human ISGs were tested for their ability to inhibit the replication of several important human and animal viruses, including hepatitis C virus, yellow fever virus, West Nile virus, chikungunya virus, Venezuelan equine encephalitis virus and human immunodeficiency virus type-1. Broadly acting effectors included IRF1, C6orf150 (also known as MB21D1), HPSE, RIG-I (also known as DDX58), MDA5 (also known as IFIH1) and IFITM3, whereas more targeted antiviral specificity was observed with DDX60, IFI44L, IFI6, IFITM2, MAP3K14, MOV10, NAMPT (also known as PBEF1), OASL, RTP4, TREX1 and UNC84B (also known as SUN2). Combined expression of pairs of ISGs showed additive antiviral effects similar to those of moderate type I interferon doses. Mechanistic studies uncovered a common theme of translational inhibition for numerous effectors. Several ISGs, including ADAR, FAM46C, LY6E and MCOLN2, enhanced the replication of certain viruses, highlighting another layer of complexity in the highly pleiotropic type I interferon system.
The interferons are a multifunctional family of around 20 cell-signaling proteins that are secreted from cells following viral infection (as reviewed here). Our cells expend a lot of energy attempting to detect infection and following this, they express high concentrations of IFN proteins. Following secretion, they bind to receptors - and activate - nearby cells alerting them to the viral assault.
Through a complex signaling network a range of genes are actively expressed across the genome that alter the cell in such a way that it becomes harder for viruses to infect them. A rapid antiviral defence system is set-up within the host's tissues and organs. As shown in mice lacking STAT1 a key IFN signal mediator, this IFN signaling network is required to limit viral replication and disease yet also bide time for the development of an adaptive immune response. IFNs are extremely important in our fight against viruses. Only a handful of these IFN-stimulated genes (ISGs) have been characterised while hundreds still sit untouched.
What did they do?
Using previously published gene expression data the group chose 389 ISGs for characterisation. To determine what function these ISGs have on virus replication, Schoggins, et al. developed an intracellular assay in which a retroviral vector expressing high levels of both the individual ISG alongside a red fluorescent protein was used to infect IFN pathway-deficient cells in vitro and then 48 - 72 hours following ISG expression, these exact cells were again infected with a range of RNA viruses expressing a green-fluorescent protein (see figure below). Viruses used included: hepatitis C virus, HIV, yellow fever virus, west Nile virus, Venezuelan equine encephalitis virus and chikungunya virus.
A) ISG/RFP-expressing retrovirus, B) experimental outline
Using basic Fluorescent Activated Cell Sorting (FACs) they were able to sort the cells based upon what colour they were, for example: red and the ISG inhibited the virus; green and it did not. More importantly they were able to specifically quantify the levels of green and red to asses... Read more »
Schoggins, J., Wilson, S., Panis, M., Murphy, M., Jones, C., Bieniasz, P., & Rice, C. (2011) A diverse range of gene products are effectors of the type I interferon antiviral response. Nature. DOI: 10.1038/nature09907
As genetic parasites, viruses use the cells within our tissues, organs and bodies to replicate. And for the most of it our cells do not like it. The more a virus replicates within a host, the more chance it will have to spread among the population yet a viral infection within a cell may set up a devastating chain of events leading to its ultimate demise.
A human brain. http://www.tehrantimes.com
For example, the virus can disrupt the structure of the inside of the cell; they can shut-down gene-expression & protein synthesis and they may even alert the immune system towards the infected "rogue-cell". These all may lead to the eventual death of the cell at the hands of either the virus or our immune system. Inflammation nor viral infection are not particularly good news for an organ. Yet most of our organs can regenerate and heal following an infection while others find this task more difficult. Infection is especially dangerous in tissues like the central nervous system (CNS) that harbor cells which do not regularly divide: post-mitotic neurons.
Your brain as a target of virus infection
I think this is a bit of an understatement but we rely upon our nervous system to co-ordinate most of what we do on a day to day basis. Yet, many viruses have evolved to use our nervous system as an appropriate tissue to replicate within, including: rabies virus, measles and mumps viruses and west nile virus. These 'neurotropic' viruses infect our neurons, accompanying glial cells and the endothelial and epithelial cells that associate with them. Infection may result in potentially fatal cell death and inflammation in the form of meningitis and encephalitis that may develop into severe - possibly permanent - cognitive dysfunction. These diseases and their causative viruses are of extreme importance currently as many established and indeed, emerging viruses infect and cause serious CNS disease in humans and other animals.
A neuron infected with a Measles virus expressing a green fluorescent protein. http://jvi.asm.org/
How then does our body prevent viruses reaching our CNS and if it fails to stop it entering, how can it prevent it replicating within endangering its necessary functions? There exists an elaborate array of barriers throughout our entire brain that limit the entry of pathogens. The major obstacles are concentrated along the blood-brain and blood-cerebral spinal fluid (CSF) barriers. Unlike in other organs these structures limit the entry and exit of large macromolecules, e.g. viruses.
Firstly, in the blood-CNS barrier the blood vessels that would usually carry the viruses during an infection into different tissues form very tight connections together. Although viruses are small, even they would find it difficult to pass through these "tight junctions". As we penetrate deeper into the brain tissue, the blood vessels themselves are encased by a network of non-neuronal cells, including: glia, macrophages and astrocytes that function to physically and biologically back-up the protection of these vessels and stop viruses entering and spreading throughout the brain. These blood vessels are also entrapped among a synthesised basement membrane of large proteins, effectively preventing the diffusion of viruses outside of the blood-stream.
The blood-CSF barrier, located at distinct sites within the brain (choroid plexus) and meninges is somewhat different in terms of structure. As it is not protected by tightly-packed blood vessels, the blood vessels have spaces between them allowing transfer of plasma generating the CSF. As a back-up, epithelial cells line this area and regulate the transfer of "cargo".
I don't think any viruses are naturally injected straight in to the brain of any animal so how then do they reach this tissue, especially as there are so many barriers preventing them from doing so?
Despite this complex defence system, viruses have evolved many strategies to enter and spread within the CNS. Some have been shown to infect the cells making up the blood vessels themselves. Infection causes them to change their biological behaviour to favour an increase in permeability thus allowing more and more viruses to escape the initial barrier. These viruses can even infect and pass through to the other side unaffected, thereby completely bypassing the barrier function.
Olfactory neurons. http://www.healingtherapies.info/
Some viruses use what is known as the 'trojan-horse' strategy by which they infect the mobile immune cells found naturally in the body - lymphocytes - which are then targeted to the CNS blood-vessels and they can pass through into the brain. Here, they release infectious virus which go on to infect nearby neurons. Furthermore, viruses such as the rabies virus can infect distant neurons of the peripheral nervous system, far away from the brain and transport themselves up through the neuronal cell body and into the brain. Many viruses have been shown to ... Read more »
McGavern, D., & Kang, S. (2011) Illuminating viral infections in the nervous system. Nature Reviews Immunology, 11(5), 318-329. DOI: 10.1038/nri2971
Western Lowland gorilla
Gorillas, Gorilla spp. are found only throughout central African rainforest where there are in total over 200,000 individuals living in the wild. Two gorilla species are recognised, split between east and west Africa with at least two sub-species recognized in both. Their numbers are rapidly decreasing with problems such as habitat loss, poaching and human war contributing greatly to a rapid reduction in their numbers. Sadly, one other factor on top of these in which these large primates must worry about is that of the transfer of infectious agents arising from humans and other animals.
Viruses are constantly being transferred between populations of animals but may not establish infection all that easily in a non-host species. Although occasionally infection will result in virus replication and significant disease and this is known as a zoonotic infection - when non-human viruses are transmitted to humans (see HIV, Influenza A and SARS-coronaviruses) and 'reverse zoonosis' when human viruses infect other animals. This pathogen transfer may be especially important when occurring in a critically endangered species such as gorillas; one recent example is that of human metapneumovirus.
EBOV effects not just humans http://turbo.indyposted.com/
We have been able to detect the effect of a number of viruses on gorilla populations including enteroviruses, adenoviruses and parvoviruses for example, although a small number are known to cause disease. In the last decade thousands of gorillas, chimpanzees and other mammals were killed through infection with the Zaire strain of ebola virus (EBOV) in the rainforests throughout the Congo basin area of central Africa. EBOV is a highly infectious and deadly RNA filovirus which causes a nearly always fatal hemorrhagic fever. The reservoir species for EBOV has been linked to central African fruit bat populations and ebola has caused hundreds of human deaths since its first recorded emergence in the 1970s. Significantly more non-human primates have been victim to ebola than humans. We can implement a number of control measures for example limiting human-ape contact especially when ill to prevent this virus transfer but this may be more difficult when humans are not involved as in the case of EBOV. There are also a number of therapeutic options available although in the case of ape infection, would be logistically impossible. One strategy we therefore must consider is that of potentially protecting these Gorilla populations through vaccination against a number of potential viral pathogens. The group VaccinApe is attempting to do just that. A volunteer consortium lead by the charity group the World Wildlife Fund, a vaccine developer Integrated Biotherapuetics and two academic institutions, the Max Plank institute for Evolutionary Anthropology and Kansas state University, VaccinApe is trying to develop an easy and effective method of vaccinating Gorilla populations in the wild. Currently in a 'proof of concept' phase, the group will lead the development of non-human primate vaccinology in order to generate a safe and reliable Ebola virus vaccine to be used through darting of individual gorillas. A large scale vaccination program may therefore afford protection of critically endangered gorilla populations against future EBOV emergences.
In light of the clinical severity of EBOV infection in humans a number of potential vaccine candidates have been developed which rely upon the generation of a protective immune response specifically to EBOV. One main candidate the group are interested in is the EBOV virus-like particles (VLPs). These are effectively non-infectious viruses lacking a viral genome but retaining virus antigenic proteins. Therefore EBOV-specific immunity will be generated against whatever EBOV proteins are found within the VLP. In early trials in macaques, this VLP strategy protected individuals against EBOV challenge although whether this could safely be transferred to gorillas isn't known.
EBOV VLP. Looks and acts antigenically like 'live' ebola. http://www.integratedbiotherapeutics.com/
Endangered wild gorilla and chimpanzee populations are at a great risk from a number of emerging viruses with the most important being EBOV. The difficulties in preventing direct transmission/therapeutic intervention have led people to consider the development of anti-EBOV vaccines. A number of candidates have already been tested and proved safe and effective in non-human primate models possibly allowing these vaccines to be transferred to gorilla population testing. It is hardly surprising that the work required to carry out such large-scale and difficult vaccination campaigns in wild gorillas in the African rain forest will be extremely difficult. The main problems include safety/efficacy testing in gorillas, physical vaccination methods and tracking anti-EBOV immunity non-invasively. Despite these difficulties, only time will tell whether the work of VaccinApe and their partners is to be supported as a worthwhile investment to save these animals.
Le Gouar PJ, Vallet D, David L, Bermejo M, Gatti S, Levréro F, Petit EJ, & Ménard N (2009). How Ebola impacts genetics of Western lowland gorilla populations. PloS one, 4 (12) PMID: 20020045
... Read more »
Le Gouar PJ, Vallet D, David L, Bermejo M, Gatti S, Levréro F, Petit EJ, & Ménard N. (2009) How Ebola impacts genetics of Western lowland gorilla populations. PloS one, 4(12). PMID: 20020045
Richardson JS, Dekker JD, Croyle MA, & Kobinger GP. (2010) Recent advances in Ebolavirus vaccine development. Human vaccines, 6(6), 439-49. PMID: 20671437
Warfield KL, Swenson DL, Olinger GG, Kalina WV, Aman MJ, & Bavari S. (2007) Ebola virus-like particle-based vaccine protects nonhuman primates against lethal Ebola virus challenge. The Journal of infectious diseases. PMID: 17940980
Bacteriophage Lambda particles - like those used in this study
Viruses are the most common organisms on this planet and also most likely the most diverse. It is therefore unsurprising how ecologically important they are in particular environments and indeed global ecological processes. Just look at the amount of papers published on the subject recently. What is preventing us from extending these studies to more environments and preventing us having a closer look at what viruses are present is our inability to study small amounts of these microorganisms. Our knowledge of global viral diversity has most likely been biased by this lack of single-virus investigations.
In order for us to study viruses at the molecular level we have to somehow get the virus. This usually requires infecting certain cells with the virus in order to take advantage of virus replication and produce more and more copies of viral particles. This means that we have to find cells that the virus infects, which is difficult for some viruses found in environmental samples where no knowledge of host is available. This problem has been encountered with bacterial species where it was found that we were only able to grow in the lab a small percent of the total microbes from a single sample. The field of bacterial 'metagenomics' (get your free pdf review here) took off when it became easier and easier to study the molecular biology of those bacteria without the need to grow them in the lab; we are now able to fully sequence bacterial genomes without the need for culturing. These developments are now being applied to virus species in a diverse range of environments.
Single Virus Genomics (SVG)
A recent study in PLoS ONE reports the development of a strategy to overcome such problems; Allen LZ et al (2011) - of the J. Craig Venter Institute - show, in a 'proof-of-principle' study, how they can now separate individual virus particles from a mixture and submit these single viruses to genomic analysis. The results of this paper are likely to enhance many areas of virology including ecology, immunology and basic biology which is probably why the group have patented the process. Although the paper didn't actually deal with environmental samples, a paper by the same group is currently in the works.
SVG strategy: seperate, agarose and PCR
What Allen et al did was make themselves a well defined mixture of bacteriophage (viruses of bacteria) particles. They filtered this suspension in order to only get the viruses in their sample. These viruses were then dyed by the attachment of fluorescent molecules to their surfaces so as they could be separated using what is known as a flow cytometry machine. This machine allowed only single virus particles to pass through and were then 'printed' onto a microscope slide in a 'high-throughput' fashion. To stabilise the particles they embedded each virus in a bead of agarose - a gel-like substance; they were also able to visualise each virus found within the agarose. Once they had separated each particle out they then used a special kind of PCR to amplify the entire virus genome inside each agarose bead. The products of this were then used for DNA sequencing and finally assembled into the whole virus genome using computational methods.
The group are currently attempting to further optimise this protocol to make it easier and more accurate especially considering that this method cannot yet be easily used for RNA viruses nor viruses about which no sequence information is known. Also, the viruses amenable to this technique would have to be pretty stable to survive the whole process. You can read about their plans for this in the paper's discussion.
A single bacteriophage particle
What does this mean for virology in general?
Virologists have generally concentrated on studying populations of viruses in their investigations; animals or cells are infected with one million infectious virus particles and the effects on host functioning are measured, for example. What this research shows is that in some cases it is entirely possible to look at the genome of individual particles and not have to rely upon mixtures of thousands, if not millions, of genetically variable viruses. The major areas of science that may benefit from this development will be those that rely on virus discovery, for example, the assessment of total virus diversity in a given environmental sample. The ability to uncover low concentrations of virus in an infectious disease context in humans may also benefit, especially if there is difficulty in growing the virus in tissue culture. Groups studying the genetic diversity of viruses can now accurately determine the exact genome sequences of individual viruses and also quantify them. This research will hence benefit a wide range of disciplines within virology in general.
Allen LZ, Ishoey T, Novotny MA, McLean JS, Lasken RS, & Williamson SJ (2011). Single virus genomics: a new tool for virus discovery. PloS one, 6 (3) PMID: 21436882... Read more »
Allen LZ, Ishoey T, Novotny MA, McLean JS, Lasken RS, & Williamson SJ. (2011) Single virus genomics: a new tool for virus discovery. PloS one, 6(3). PMID: 21436882
The process in which a virus causes disease and dysfunction within its host is termed viral pathogenesis; the study of which is pretty important if we are to fully understand infection, replication and transmission of pathogens as well as to develop effective antivirals and vaccines. Ebola virus (EBOV) is one such deadly virus in which there are currently no approved antivirals nor vaccines and which the study of pathogenesis is therefore ever more important.
EBOV particle. www.accessexcellence.org
First isolated in the late '70s, EBOV now causes significant epidemics occurring with increasing frequency - the latest in early 2009. It is believed that bats play an important role in the natural replication cycle of these viruses and hence may transmit it to neighboring human and other animal populations. There are currently four 'species' of EBOV recognised which infect humans: Zaire, Sudan, Ivory Coast and Bundibugyo - yet a fifth related virus, EBOV-Reston is known only to infect non-human primates in captivity and domesticated pigs. These viruses which are mainly found throughout Central and Western Africa cause a hemorrhagic fever in humans which most often or not leads to death; EBOV-Zaire (ZEBOV) is the most deadly of all types resulting in at most 90% of those infected to die; the reasons for such high mortality are not known.
We can begin to build up a picture of ZEBOV infection and pathogenesis using a combination of in vitro studies, animal models and clinical work carried out in humans. In animal models EBOV has been shown to replicate to extremely high levels, induce the abundant secretion of inflammatory signalling molecules, cause massive cell death of lymphocytes and a great deal of tissue destruction eventually leading to multi-organ system failure, toxic shock and death. Work carried out on blood samples collected from both human survivors and non-survivors of EBOV outbreaks have showed that lethal infection is associated with a highly deregulated immune response. Extremely high levels of pro-inflammatory cytokines (cell signalling proteins) were detected as well as low levels of circulating T lymphocytes. Yet there was a surprisingly little anti-viral response seen. This all suggests that the EBOV induces a rapid and strong pro-inflammatory immune response without eliciting effective innate and adaptive antiviral defenses resulting in a lethal outcome. Those people which did not develop this lethal immune reaction survived EBOV infection. But how exactly does EBOV do this?
New evidence has been uncovered suggesting that certain ZEBOV components may act like what are known as 'superantigens' (sAg) which may responsible for this damaging deregulation in immune responses.
Superantigen TCR/MHCII interaction. anagen.ucdavis.edu
As the paper explains:"SAgs are microbial proteins that bind simultaneously to major histocompatibility complex class II molecules and to the T-cell receptor (TCR) V beta region. This “bridge” skews the T-cell repertoire by amplifying specific T- cell V beta subsets, which then are either rapidly deleted by activated cell death or become anergic."What this means is that maybe ZEBOV proteins are able to bind to our own immune cells through an abnormal mechanism (TCR AND MHCII) which results in those cells going into overdrive and then being selectively killed by our bodies (in order to prevent a runaway immune response) or those cells becoming immunologically unresponsive (anergic). If EBOV did encode a sAg it may be able to vastly eliminate our adaptive immunity.
Vbeta region mRNA expression levels in ZEBOV infected patients
A major feature mentioned above is the massive levels of cell death in T lymphocyte populations. There are many ways in which EBOV could produce this effect, including up regulation of pro-cell death molecules inside lymphocytes or through an alternative indirect pathway via infection of dendritic cells and macrophages - as it is known EBOV infects these cells. These immune regulatory cells are responsible for controlling the levels of other immune cells produced (they are considered major immunoregulators)- alter their behavior and you alter the number of lymphocytes. The finding the EBOV may encode a superantigen adds to the growing number of ways this virus may inhibit host immune responses.
We can begin to investigate this by looking at the specific types of T cell found within EBOV infected patients - normally you would expect each T cell V region type to found to the same level. sAG activity leads to the loss of specific T cell types without loss of others. Looking at the expression levels of specific V regions of the TCR we can observe whether certain types decrease after being infected with EBOV. This recent report demonstrated just this in infected patients versus non-infected (see above) suggesting that something in EBOV may act as an sAg. This appears to correlate well with a fatal immune response.
Of course we will have to further verify this finding possibly in animal models or cell culture work but we can begin to ask further questions: does this occur in other EBOV species? What protein is causing the sAg activity? Can we somehow inhibit its activity? And, how is it that some patients come to avoid a sAg response?
Leroy EM, Becquart P, Wauquier N, & Baize S (2011). Evidence for ebola virus superantigen activity. Journal of virology, 85 (8), 4041-2 PMID: 21307193
... Read more »
Wauquier N, Becquart P, Padilla C, Baize S, & Leroy EM. (2010) Human fatal zaire ebola virus infection is associated with an aberrant innate immunity and with massive lymphocyte apoptosis. PLoS neglected tropical diseases, 4(10). PMID: 20957152
2011 may be the year where the last known officially acknowledged stocks of the deadly smallpox virus, variola are destroyed - a virus that claimed over 500 million lives in the 20th century alone. The extensive collection of 'live' virus and DNA stocks totalling over 500 isolates/strains, which are held between the US Centres for Disease Control and the Russian State Research Centre of Virology and Biotechnology may be ordered to be eliminated following World Health Organisation (WHO) recommendations soon to be announced.
Although the impending fate of this pathogen has been covered elsewhere by Vincent Racaniello and Steven Salzberg I have been led to ponder its beginnings, at least in humans: where and when, over the course of human history did variola virus emerge and have we always suffered from it? What confuses the matter further is that there are two clinical forms of smallpox - major (30% mortality) and minor, including both African and Alastrim minor (<1% mortality)- do these viruses have the same evolutionary history and if so, when and where did they diverge? Luckily, we can now study the origins of infectious diseases through both molecular and historical records.
A depiction of Shapona the west-African Yoruba god of smallpox. Courtesy James Gathany (photo), CDC/ Global Health Odyssey.
Conflicting historical records
It has been very confusing trying to make sense of the historical records of suspected smallpox cases as there are significant gaps in documentation and many conflicting reports. Smallpox-like skin lesions have been observed on Egyptian mummies dating from as far back as 1580 B.C yet there is no mention of the disease at all in the Old or New testaments nor even the Hippocratic texts. There was some mention of a smallpox-like disease in China and India as early as 1500 B.C but the only unmistakable description can be found from the 4th century A.D in China. Interestingly there was no mention of smallpox in the American continents nor in sub-Saharan Africa prior to European exploration. But as shown in the picture above, smallpox has shaped west-African culture. So, did smallpox originate in Asia and spread to Egypt around 1,500 B.C? Or, is smallpox a relatively recent human disease, emerging around the 4th century A.D in Asia?
Molecular data shed light on variola evolution
A 2007 study using genomic data from the CDC's variola collections - the same ones that may soon be destroyed, added a phylogenetic perspective to the origins of smallpox and how it spread worldwide. Through studying single nucleotide polymorphisms (SNPs) from 47 variola genome isolates from geographically distant areas and collected between the 1940s and '60s they examined the genetic relatedness between isolates and were able to estimate the time since they shared a last common ancestor. They combined this DNA evidence with the above historical records to generate an idea as to where, when and how smallpox originated and spread throughout human populations.
Variola genome phylogeny
Abstract: Human disease likely attributable to variola virus (VARV), the etiologic agent of smallpox, has been reported in human populations for >2,000 years. VARV is unique among orthopoxviruses in that it is an exclusively human pathogen. Because VARV has a large, slowly evolving DNA genome, we were able to construct a robust phylogeny of VARV by analyzing concatenated single nucleotide polymorphisms (SNPs) from genome sequences of 47 VARV isolates with broad geographic distributions. Our results show two primary VARV clades, which likely diverged from an ancestral African rodent-borne variola-like virus either ≈16,000 or ≈68,000 years before present (YBP), depending on which historical records (East Asian or African) are used to calibrate the molecular clock. One primary clade was represented by the Asian VARV major strains, the more clinically severe form of smallpox, which spread from Asia either 400 or 1,600 YBP. Another primary clade included both alastrim minor, a phenotypically mild smallpox described from the American continents, and isolates from West Africa. This clade diverged from an ancestral VARV either 1,400 or 6,300 YBP, and then further diverged into two subclades at least 800 YBP. All of these analyses indicate that the divergence of alastrim and variola major occurred earlier than previously believed.
Hypothesised spread of variola worldwide
When analysed, variola fell into two large monophyletic clades signifying a historical divide in their genetic relatedness. The earliest representative - or most basal - of the variola major smallpox viruses are the Asian isolates. This suggests that major may have originated in Asia followed by geographic radiation across the Old world and into Africa. Using historical records as a means to calibrate variola evolutionary history, their results indicated that smallpox spread from Asia as much as 1,600 years ago which neatly backed up the historical records of 4th Century China. By the time smallpox reached out of East-Asia, the ancient Greek and Roman civilisations were no more - hinting that the reason they didn't observe smallpox was because at that time, in the Mediterranean region there wasn't any variola virus transmission. Despite this, analysis of the second major clade suggested a split 6,300 years ago placing variola well into ancient history. So, is smallpox a very old or relatively recent human pathogen? And, if so, where did it occur? The molecular data also showed that the clinically 'minor' forms of smallpox - African minor and Alastrim minor are very much related to the major viruses; evolutionarily speaking these viruses are thus very smilier.
A rodent origin of smallpox?... Read more »
Gubser C, & Smith GL. (2002) The sequence of camelpox virus shows it is most closely related to variola virus, the cause of smallpox. The Journal of general virology, 83(Pt 4), 855-72. PMID: 11907336
Li, Y., Carroll, D., Gardner, S., Walsh, M., Vitalis, E., & Damon, I. (2007) From the Cover: On the origin of smallpox: Correlating variola phylogenics with historical smallpox records. Proceedings of the National Academy of Sciences, 104(40), 15787-15792. DOI: 10.1073/pnas.0609268104
Raymond S. Weinstein. (2011) Should Remaining Stockpiles of Smallpox Virus (Variola) Be Destroyed?. Emerg Infect Dis, 17(Apr). info:/10.3201/eid1704.101865
Rimoin AW, Mulembakani PM, Johnston SC, Lloyd Smith JO, Kisalu NK, Kinkela TL, Blumberg S, Thomassen HA, Pike BL, Fair JN.... (2010) Major increase in human monkeypox incidence 30 years after smallpox vaccination campaigns cease in the Democratic Republic of Congo. Proceedings of the National Academy of Sciences of the United States of America, 107(37), 16262-7. PMID: 20805472
Dengue virus, DENV - an important mosquito-borne virus
Arthropods are important vectors in the transmission of a number of animal and human pathogens. A major vector group are the mosquitoes of which there are over 3,000 species. However, during their life cycle some mosquitoes feed on the blood of other animals - creating an excellent chance for the direct transfer of manymicrobial species. From here the bacteria/viruses/parasites can initiate infection of the new host which then, following another blood feed, may transmit the pathogen to an uninfected insect. This particular lifestyle allows for the development of pathogen control strategies aimed at interfering with the vector species. If we remove or inhibit the vector, we may prevent the spread of the pathogens they carry.
Another group of mosquito-borne pathogens are the closely related - but distinct - dengue viruses (DENV). DENV is commonly responsible for a 'flu-like- illness' in humans but complications may include a potentially fatal hemorrhagic fever. The incidence is mainly constrained to tropical and sub-tropical areas although in recent decades it has spread to other areas where it may cause massive epidemics. The World Health Organisation states that, "Some 2.5 billion people – two fifths of the world's population – are now at risk from dengue. WHO currently estimates there may be 50 million dengue infections worldwide every year." There is currently no commercially licensed vaccine or antivirals available for the treatment of dengue leaving the only option to prevent transmission through control of mosquito populations.
The most important vector species is the predominantly urban species Aedes aegypti - control of which may aid DENV eradication. There are a number of potential strategies that could be employed to reduce the numbers of this species including chemical poisoning, genetic strategies and biological control. A major goal is therefore to inhibit mosquito reproduction and feeding behaviour yet this requires intimate knowledge of mosquito reproductive biology. Insects communicate via a number of chemical signals, one mode of communication is via the males ejaculate -what happens to be a convenient opportunity to control a females behaviour. A mated female is behaviourally very different to an unmated one and there is evidence suggesting that this change is initiated by the transfer of male-derived signaling proteins during mating. It may then prove to be useful to identify some of these molecules so as to possibly control mosquito behaviour ourselves.
What is so good about male semen?
Sirot et al recently report, using proteomic analysis, the identificantion of a number of proteins, termed seminal fluid proteins (Spfs) transferred from males (with labelled proteins) to females (non-labelled proteins) during mating. Of which some may be responsible for the male control over female post-reproductive behaviour; this they say, lays the groundwork for future studies investigating the molecular mechanisms behind how they work and their potential use in controlling vector populations. However, care must be taken in interpreting these results as this study does not directly look at the biological effects of these proteins and does not prove that they do have any effect on female behaviour.
Labelled insect sperm
So, what do these semen proteins actually do?
Using this approach they identified 145 proteins transferred from males to females, 17 of which were previously unknown to science - 93, they say, could be assigned as potential biologically active proteins. What function do this proteins have? Well, based on the previous annotation in protein databases, they were able to assign each of their proteins a potential function indicating the potential important roles in female behaviour. These proteins are predicted to be involved in their reproductive biology, specifically protein degradation and hormonal signalling.
This work has identified a number of proteins present in the semen of the dengue vector, Aedes aegypti which are transferred from males to females during mating. This may mean they are involved in the control of female behaviour. Although this work did not look at the function of any proteins directly, it does lay the foundations for future studies. Researchers may now focus there investigations on a set of a now verified smaller set of proteins and genes. This species is also the vector for a number of other viruses such as chikungunya and yellow fever and so any work on this may aid the control of these important diseases. Gillott C (2003). Male accessory gland secretions: modulators of female reproductive physiology and behavior. Annual review of entomology, 48, 163-84 PMID: 12208817
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Gillott C. (2003) Male accessory gland secretions: modulators of female reproductive physiology and behavior. Annual review of entomology, 163-84. PMID: 12208817
Sirot, L., Hardstone, M., Helinski, M., Ribeiro, J., Kimura, M., Deewatthanawong, P., Wolfner, M., & Harrington, L. (2011) Towards a Semen Proteome of the Dengue Vector Mosquito: Protein Identification and Potential Functions. PLoS Neglected Tropical Diseases, 5(3). DOI: 10.1371/journal.pntd.0000989
Daily, our immune system deals with multiple microbial threats, including viral, bacterial, fungal and parasitic pathogens that have evolved to evade our defences. One major obstacle to infection is our 'innate' immune system - the one that doesn't include all our B and T cells; has no memory and is generally pretty fast in acting. This set of barriers is made of anatomical, chemical, molecular and cellular obstacles that must be overcome if a pathogen is to successfully set up home in our bodies.
Activation results in caspase-1
recruitment and proteolysis
This system has been studied for decades, yet relatively recently, a novel component was discovered - the inflammasome. On top of bein implicated in protecting against microbial pathogens it has also been shown to play a role in many autoinflammatory diseases, such as inflammatory bowel diseases and vitiligo. It is beginning to emerge as a central component in the regulation of our defences.
PAMPs, DAMPS and PRRs
Detected in many of our cells, the inflammasome is a multi-protein complex whose assembly is triggered by the detection of what are known as 'pathogen-associated molecular patterns' (PAMPs) - basically something our cells can detect which looks only like bacteria, virus, fungi or parasite. These PAMPS are sensed by cellular proteins known as 'pathogen recognition receptors' - (PRRs) e.g NALP3, on the inside or outside of the host cell; however, triggering can also result from the detection of chemical 'danger-associated molecular patterns' (DAMPS) compounds, including uric acid and asbestos. PRRs include: double stranded DNA, non-capped RNA and lipopolysacharride found in bacterial cell walls. Triggering of other PRRs results in the activation of many other immune responses, including autophagy, interfon secretion and cell signalling.
A pro-inflammatory signalling molecule
Formation of the inflammasome results in the activation of multiple pathways responsible for co-ordinating our immune response, yet interestingly, there are multiple forms of inflammasomes made up and triggered by different sets of proteins. This initial step of activation has been covered very well before, here. The activated inflammsome goes on to trigger key downstream members of our innate immune system through the recruitment of an important regulatory protease (it cuts up other proteins) - caspase 1, which converts inactive molecules to active, pro-inflammatory ones, such as interleukin-1 beta and interleukin-18. This 'inflammatory cascade' functions to initiate an effective local and systemic immune response through the control of the innate and adaptive immune system; for example, IL-beta is responsible for fever and the recruitment of immune cells to the site of infection, and IL-18 induces the development of key T cell responses.
Not all IL-1beta and IL-18?
Recent studies have shown that not all immune functions of the inflammasome are down purely to the effects of these two mediators; a number of other effectors are implicated in our complicated immune responses. Other responses activated by caspase-1 proteolysis include the better cell survival in the face of bacterial toxins; regulation of cellular metabolism limiting pathogen replication; induction of pyroptosis - a pro-inflammatory form of cell-death much like apoptosis and the secretion of high levels of multifuctional cytokines from the cell.
Inflammatory 'pyroptosis' is looking to be an important mediator in the immune response
All in all, the inflammasomes represent a fast and effective barrier to microbial infection in eukaryotic cells which, following detection of PAMPS and subsequent activation, results in a powerful innate immune response, stimulation of adaptive defences and significant contributions to over-all host defence using a variety of pathways.
Inflammasomes and viruses
As the cell is able to detect pathogens (PRRs and PAMPs) and assemble the inflammasome to regulate the immune response, it is clearly in the best interest of the virus (basically a bag of PAMPs) to evade or somehow counteract its effects through preventing its formation or inhibiting its ability to activate an immune response. We are, however, only beginning to understand the complex relationship viruses have with the inflammasome through investigating how viruses trigger its assembly; how viruses trick the inflammasome into failing to act and how triggering of the inflammasome relates to the particular disease symptoms a virus causes. How does this system specifically detect viruses? What is the response to pathogens? And, what role does this play in host immunity?
Hoffman HM, & Brydges SD (2011). The genetic and molecular basis of inflammasome-mediated disease. The Journal of biological chemistry PMID: 21296874
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Hoffman HM, & Brydges SD. (2011) The genetic and molecular basis of inflammasome-mediated disease. The Journal of biological chemistry. PMID: 21296874
Kanneganti, T. (2010) Central roles of NLRs and inflammasomes in viral infection. Nature Reviews Immunology, 10(10), 688-698. DOI: 10.1038/nri2851
Martinon F, Burns K, & Tschopp J. (2002) The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-beta. Molecular cell, 10(2), 417-26. PMID: 12191486
In order to better understand (and maybe even enjoy - is that the right word?) viruses, I think it is time to meet and greet a couple of them; this is why I am starting a weekly tradition here at Ro6 - "Virus of the week". I will introduce a virus; discuss its importance; highlight the important aspects of its biology and how it infects its hosts.
The first virus shown here is one currently close to my heart: the mumps virus (MuV). As part of my PhD work, I am using this virus to understand its molecular biology and how it causes disease in its host - humans. Not much is known about the basic biology of this unique human pathogen but that is beginning to change.
What is mumps?
Mumps, what used to be a common childhood illness, is characterised by the swelling of the salivary glands (parotitis) following infection yet something not commonly known is that it can also very easily infect the central nervous system with rates nearing 50% of those with mumps (see the NHS info site here). Prior to the introduction of vaccination programs it was one of the biggest causes of non-bacterial meningitis worldwide, although not life-threatening, meningitis and encephalitis constitute serious disease. Also, very importantly, in males it is very common for the testis to swell massively causing great pain and discomfort, possibly leading to reduced fertility. There are also a number of much rarer complications associated with MuV infecton, including infection of the heart muscle; the pancreas and the inner ear which can lead to deafness (Lancet review here). Part of my research is trying to understand why - at the molecular level - MuV causes these symptoms and infects these organs rather than others.
Why is it important?
The mumps virus
But why is it important to study MuV, especially as we have a good vaccine? Well, despite this highly effective vaccine - that HAS worked very well in the past - mumps is still currently circulating worldwide. A good example is the ongoing Scottish outbreak but well publicised outbreaks across the US and UK also highlight this. The reasons why, despite being vaccinated, MuV can infect a person are currently unknown. If we have a good vaccine and the ability to vaccinate people easily yet can't eradicate the mumps virus, what hope do we have for a virus which is more difficult to immunise? Also, in countries lacking mumps immuisation, mumps can contribute to profound morbidity and mortality; mumps is therefore still a global problem! For WHO figures see here.
Mumps virus molecular biology
Viruses are, at the most basic level, very small packages of nucleic acids and proteins packaged in a protective coating which can either be made of proteins (capsid) or fats (an envelope). The mumps virus comes from a group of viruses that whose genome is made out of RNA - not DNA like you and I have - and strangely, it is also only single-stranded while ours is double-stranded; we say that this RNA is 'negative sense' in that it is complementary to its mRNA molecules. The mumps genes are lined up across this single RNA molecule encoding for proteins involved in cell entry, replication and immune evasion.
Other viruses with single-stranded, negative-sense RNA genomes include many important human and animal pathogens: measles virus, respiratory syncitial virus, ebolavirus and the henipaviruses. More specificaly MuV is a Rubulavirus found within the family Paramyxoviridae. Like other viruses, MuV carries out its entire active lifecycle within the cell and uses its extracellular form (shown above) to infect new cells allowing the virus genome access to the cell cytoplasm where replication and gene expression take place and full-blown infection can begin.
Evolutionary tree of Paramyxovirus relationships - notice mumps virus (MuV), a rubulavirus.
So how does the mumps virus cause mumps?
Just like Influenza, mumps is transmitted through aerosol particles that we breathe in and out which allows the virus access to our respiratory tract - a very nice point of entry of many viruses. MuV is believed to first replicate along our airways in the epithelial cell lining and then is somehow able to spread throughout the body, possibly via infection of your bodies immune cells or maybe just release of virus particles into the bloodstream. Infection of the salivary glands, brain, testis and pancreas follows giving rise to the common symptoms known as mumps. Virus particles are finally released back into your airways allowing the spread between person to person and once your immune response kicks in, the virus infection is cleared up hopefully without any long-term problems.
What does the future hold for mumps research?
A lot of this basic knowledge outlined above on mumps infection has been seen only through studying of other viruses and there is so much more basic mechanisms to find out. The advent of modern molecular biology techniques and protocols has allowed for a renewed interest in studying this basic biology of viruses, including mumps. The ability to alter a virus genome and investigate whether it has biologically changed has facilitated a much better understanding of viral infection, replication and evolution. This has - and is being - applied to the study of mumps, however, there is still much work to be done to catch up with other viruses. Maybe, before we have had a chance to understand mumps biology we will have eradicated indigenous mumps transmission worldwide and it will no longer be important.
Galazka AM, Robertson SE, & Kraigher A (1999). Mumps and mumps vaccine: a global review. Bulletin of the World Health Organization, 77 (1), 3-14 PMID: 10063655
... Read more »
Walker J, Huc S, Sinka K, Tissington A, & Oates K. (2011) Ongoing outbreak of mumps infection in Oban, Scotland, November 2010 to January 2011. Euro surveillance : bulletin europeen sur les maladies transmissibles , 16(8). PMID: 21371413
Mammals hold a large reservoir of potential emerging viruses
Viruses universally infect the tree of life; all species ever known have most likely acted as hosts to viral pathogens. These species, harbouring many unknown viruses may therefore act as virus ‘reservoirs’ – storing potential emerging viruses that may one day infect humans or other species. This is illustrated by the recent emergences of swine and avian-origin Influenza, SARS-coronavirus and the Henipaviruses. Events just like these have probably contributed multiple viral pathogens to the human species over our evolutionary history, for example: HIV and the measles and mumps viruses.
Being able to predict when and where these events may take place is thus important to global health if we are to prevent further deadly pandemics. We therefore must be able to accurately judge zoonotic risks before they happen. Many processes, such as genetic factors, contact between species and the amount of virus present in the population will affect the emergence of viral pathogens; if we are to track these it may therefore be possible to predict these risks easily.
The European 'greater mouse-eared bat', Myotis myotis
Possibly due to them being relatively close relatives to us, mammals constitute the largest threat in terms of future viral pathogens that we know of. The most abundant mammals are the rodents and bats – making up well over 50% of all known mammal species with their large population sizes and geographic closeness to human populations favouring for the frequent ‘spill-over’ of viruses to us. It is therefore not surprising that many emerging viruses have originated in these species. Bats have gained much attention from the scientific community relating to virus emergence and have been shown to harbour multiple RNA and DNA viruses, although the extent to which each may spread to humans and other species is unknown (See recent metagenomic papers 1 and 2).. Despite the international awareness of bats as virus reservoirs, little work has been carried out investigating both the molecular biology of bat viruses and even the epidemiological and ecological dynamics of viruses within bat populations. If we are to fully understand how best to prevent virus emergence from bats and other species we will have to fully investigate these processes.
Drexler et al(2011) recently investigated the epidemiological dynamics of viruses in a single population of Myotis myotis bats in Germany in order to better understand the role these animals play in virus ecology.
Bats host noteworthy viral pathogens, including coronaviruses, astroviruses, and adenoviruses. Knowledge on the ecology of reservoir-borne viruses is critical for preventive approaches against zoonotic epidemics. We studied a maternity colony of Myotis myotis bats in the attic of a private house in a suburban neighborhood in Rhineland-Palatinate, Germany, during 2008, 2009, and 2010. One coronavirus, 6 astroviruses, and 1 novel adenovirus were identified and monitored quantitatively. Strong and specific amplification of RNA viruses, but not of DNA viruses, occurred during colony formation and after parturition. The breeding success of the colony was significantly better in 2010 than in 2008, in spite of stronger amplification of coronaviruses and astroviruses in 2010, suggesting that these viruses had little pathogenic influence on bats. However, the general correlation of virus and bat population dynamics suggests that bats control infections similar to other mammals and that they may well experience epidemics of viruses under certain circumstances.
Following the extraction of nucleic acids from bat droppings collected every 3 weeks in May, June and July over three years they were able to build up a generalised picture of how the number and diversity of specific virus genome sequences changed over time and how this may influence the bat population. Using this method, they detected a total of 7 separate viral sequences from a single coronavirus, 6 astroviruses (both RNA viruses) and one adenovirus (DNA). From this, they were able to track their abundance over the 3 months noting whether the amount of a virus increased or decreased over time. Despite there being no evidence that these viruses cause disease in humans or other animals, these may be used as a proxy for other deadly pathogens, including SARS-coronavirus and ebolavirus. A significant increase in bat numbers over the study period indictaed that these viruses had little pathogenic influence on populations.
Virus sequence abundance over the 3 years. A = coronavirus. B = Astrovirus and C = Adenovirus. Notice the cyclical dynamis with A and B yet not C
Two general patterns emerged from these studies:
One where abundance initially increased only to decrease soon after and finally, in the last month increasing again – shown with the coronavirus and astroviruses. They attribute these different patterns to fundamental changes within the bat population. The initial increase in virus abundance (coronavirus and astrovirus) is possibly due to the increase in the number of bats living in the colony – as time goes on, the colony expands taking in new bats which are susceptible to virus infection just as is seen in human populations. As the bats become immune to the viruses and begin to give birth to pups of their own the transfer of maternal protective immunity to newborns causes an initial decrease in virus numbers; immunity to the virus is still present and hence abundance decreases. As maternal protection fades over time, the newborns become susceptible to infection, leading to rapid increases in virus abundance. These cyclical dynamics a... Read more »
Drexler JF, Corman VM, Wegner T, Tateno AF, Zerbinati RM, Gloza-Rausch F, et al. (2011) Amplification of Emerging Viruses in a Bat Colony . Emerg Infect Dis, 17(3). info:/10.3201/eid1703.100526
How can you tell how safe a vaccine is?
Mumps, a highly infectious viral disease, has been largely eradicated in the developed world following the introduction of a highly effective live-attenuated vaccine. Highlighted by well-publicized outbreaks in the U.S and U.K, the number of cases, however, has risen causing worldwide alarm. The reasons for this re-emergence have yet to be fully elucidated but most likely are due to a number of factors, including waning immunity and poor vaccine coverage.
Despite what is normally reported, mumps infection can cause serious complications. Prior to the introduction of the vaccine - and of course in countries that fail to administer it - mumps infection was/is the most common cause of viral meningitis and encephalitis; it has been estimated that 50% of those infected by mumps have some form of central nervous system involvement, although 1 - 10% will actually experience a symptomatic infection. It is safe to say that the mumps virus is one of the most neurotropic human viruses currently circulating and that its neurotropism can hardly be considered a complication.
All this really underlines the importance of maintaining mumps vaccination in protecting individuals and populations from serious disease. The key then is to develop not just more effective vaccines but also safer vaccines as people aren't likely to give their children a vaccine which may cause serious side-effects especially considering the propensity for mumps virus to cause CNS disease. A recent review of mumps vaccine safety states that,
Such a problem places public confidence in all mumps vaccines at risk, as indicated by the experience in Japan where national mumps vaccination programs were discontinued in 1993 following established links to aseptic meningitis; consequently, more than a million new mumps cases occur annually in that country
The cotton rat - is this the future of mumps vaccine safety?
How then are we to assess the safety and more specifically 'neurovirulence' of mumps candidate vaccine stocks? Recently, Rubin and Afzal from the United States Food and Drug Administration and the UK National Institute for Biological Standards and Control respectively, outlines the current state of the art in mumps virus safety testing and outlines how its future might look. What we would like in a test system is for it be accurate and fully predictive (limit false positives and negatives); it would need to economical (vaccines need a lot of testing) and it needs to be relatively easy to carry out and replicate. For us to do this, these methods require vigorous testing!
Currently, much like other virus vaccines, mumps vaccine safety is assessed in a monkey model and has resulted in the detection of significantly attenuated vaccines for use in humans. There is however cause for concern with this system as in some instances it fails to distinguish between important differences in levels of attenuation. There is therefore a need to replace this system if not on the grounds of ethical and economic concern but on the grounds of safety. In has stepped a small animal model - the cotton rat- which has been shown to better predict neurovirulence; is cheaper and is less ethically taxing; it is hence subject to a WHO validation study.
False colour electron micrograph of the mumps virus
But why do we have to use animal models at all for safety testing? Can we not just be content with in vitro studies with cell lines? In some cases, we can predict how a virus will act within an animal on the basis of studying how it infects and replicates in cell line but there is, however, no in vitro alternatives for mumps - at least not yet - and even if there were we can't say whether it could ever fully replace animal studies.
In some systems, animal infections just cannot be replaced if we are to maintain a high level of vaccine safety which of course is important when vaccines are administered to billions of people worldwide we are then forced to stick with animal testing. We can rest assured that with recent developments in small-animal models, future testing may come more accurate, cheaper and a little more ethically pleasing.
BRUYN HB, SEXTON HM, & BRAINERD HD (1957). Mumps meningoencephalitis; a clinical review of 119 cases with one death. California medicine, 86 (3), 153-60 PMID: 13404512
Dayan GH, & Rubin S (2008). Mumps outbreaks in vaccinated populations: are available mumps vaccines effective enough to prevent outbreaks? Clinical infectious diseases : an official publication of the Infectious Diseases Society of America, 47 (11), 1458-67 PMID: 18959494
Rubin, S., & Afzal, M. (2011). Neurovirulence safety testing of mumps vaccines—Historical perspective and current status Vaccine DOI: ... Read more »
BRUYN HB, SEXTON HM, & BRAINERD HD. (1957) Mumps meningoencephalitis; a clinical review of 119 cases with one death. California medicine, 86(3), 153-60. PMID: 13404512
Dayan GH, & Rubin S. (2008) Mumps outbreaks in vaccinated populations: are available mumps vaccines effective enough to prevent outbreaks?. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America, 47(11), 1458-67. PMID: 18959494
Rubin, S., & Afzal, M. (2011) Neurovirulence safety testing of mumps vaccines—Historical perspective and current status. Vaccine. DOI: 10.1016/j.vaccine.2011.02.005
In order to survive and replicate within their hosts, viruses must manipulate those pathways and systems in which their host relies upon for its own survival. However, this model gets more complicated with those viruses successfully infecting multiple host species. For example, Dengue virus (DENV) – an emerging pathogen which causes over 50 million cases a year of a mild to deadly disease – infects both humans and mosquito species of the Aedes genus. Thus to accomplish survival, DENV must interact with proteins from these two distantly related hosts. Given this complexity, understanding this dual-host/pathogen system is considerably difficult yet as Doolittle and Gomez (2011) show, computational approaches based on structural predictions of viral and host proteins may allow for the accurate prediction of the complex in vivo ‘interactome’.
Transmission of DENV - the principle mode is direct mosquito to human
The group set out to understand the interactions between both DENV encoded proteins and those of its hosts – humans and Aedes mosquitos. Using previously determined structural information for human and fly (relatively closely related insect to Aedes) and how these proteins interact with each other, they were able to map these back on to host infection. They searched databases for structural similarities between dengue proteins and those from its host (human or fly) – these ‘dengue similar host proteins’ were used to search for host-host protein interactions as a surrogate for host-dengue interactions.
“The computational methodology employed to generate this map assumes that proteins with comparable structures will share interaction partners. Therefore, we predict that DENV2 proteins may merge into the host protein interactome at the points normally occupied by structurally similar host proteins, creating an interface for the manipulation of downstream host processes.”
Using this approach, they built up a network of possible host/pathogen interactions, assuming that DENV proteins can participate in the same interactions as host proteins. Of course, this method over estimates interactions so to counter this, they prioritised particular interactions for further study based on previously published, validated in vivo work and those interactions still left hopefully were functionally accurate and important. This approach had previously been used to study human-HIV-1 protein interactions.
DENV capsid structure
Following significantly limiting their map down to those that had been previously validated the biological functions of host target proteins and dengue-similar proteins were analysed to determine whether the predicted functions matched those that would be important for viral infection in both humans and mosquitoes. As shown above, DENV-like proteins participate in interactions involved in diverse processes – importantly including cell death, signalling cascades, immune response and metabolism. They focus the investigation into DENV manipulation of host apoptosis and innate immune signalling and also those proteins which are shared between both insect and human hosts.
They suggest that due to the disparity in the known molecular biology of dengue/host interactions this computational methodology has its limitations in this system yet these data should be used as a springboard for future investigations and hypotheses. This study highlights the importance of global computational analysis in determining basic host/pathogen biology especially in a system which has been poorly studied like DENV.
Doolittle, J., & Gomez, S. (2011). Mapping Protein Interactions between Dengue Virus and Its Human and Insect Hosts PLoS Neglected Tropical Diseases, 5 (2) DOI: 10.1371/journal.pntd.0000954
Dyer MD, Murali TM, & Sobral BW (2007). Computational prediction of host-pathogen protein-protein interactions. Bioinformatics (Oxford, England), 23 (13) PMID: 17646292... Read more »
Doolittle, J., & Gomez, S. (2011) Mapping Protein Interactions between Dengue Virus and Its Human and Insect Hosts. PLoS Neglected Tropical Diseases, 5(2). DOI: 10.1371/journal.pntd.0000954
Dyer MD, Murali TM, & Sobral BW. (2007) Computational prediction of host-pathogen protein-protein interactions. Bioinformatics (Oxford, England), 23(13). PMID: 17646292
In the modern world, we are continuously challenged by viral disease; well established pathogens such as the measles and mumps viruses alongside recently (re)-emerging viruses such as ebola-virus and even those viruses which we currently know little about (XMRV?) all represent a continuous threat to human health and well-being. Yet how can this be true when we have been developing anti-viral vaccines for half a decade – surely we should be good at it by now? And, is this idea that we can easily eradicate viruses hollow, considering how relatively easy it may be to recreate long extinct pathogens from their nucleic acid sequence alone? These investigations will require years of research and billions of dollars in funding but how should it achieved?
NEIDL building in Boston
In a recent article (UK Society for General Microbiology publication 'Microbiology Today', can be found here), Paul Duprex and Elke Mühlberger
, both virologists from the Boston UniversitySchool of Medicine and associated National Emerging Infectious Disease Laboratories (NEIDL) put forward their view on how best virology may be able to face up to this global challenge and outline how it may be achieved. Through a firm grasp of its historical context, combined with recent developments in molecular biology, future scientists will better be able to understand the intertwined relationship between viral pathogenesis and its rational attenuation. If we understand how viruses cause disease at the molecular level, altering this through well-established DNA technologies we may be able to mitigate pathogenesis and develop improved or novel vaccines – and antivirals - on a rational level.
In the early days of virology (see site on the history of vaccines), bent on developing vaccines under the paradigm of “isolate, attenuate and vaccinate”, scientists barely understood the mechanisms behind the production of live-attenuated vaccines, such as those for measles and smallpox. They didn’t need to; they worked superbly and were of course highly effective allowing for the eradication of one of the worst diseases of mankind. But this golden age didn’t last long, with countless viruses proving somewhat more resistant to this ‘black box’ method of vaccinology; HIV-1, SARS and Ebola had not yet been observed by scientists and nothing was known about them. This was an age concentrated on investigating viral pathogenesis and how best to change it but with the developments of recombinant DNA methodology (two important papers concerning virus cloning and synthetic virology: 1 and 2) this agenda shifted in favour of the virus genome and it is hard to even outline the tremendous impact this molecular understanding of viruses has had on both basic and applied virology. Yet bear in mind that it is this same technology that could facilitate the resurrection and recreation of ‘eradicated’ virues.
Knowledge of the molecular biology of viruses (in this case measles virus) will go a long way in developing much needed novel, rational vaccines
Despite this word of caution, Duprex and Muhleberger argue that virology has – or at least should – come back full circle, back to understanding basic pathogenesis with the aim in mind of developing more effective therapies and vaccines; this, they say, is needed now more so than ever. This generation, and the next, of molecular virologists should take heed of the long historical roots their discipline has and highlight the importance of understanding disease and attenuation as two sides of the same coin. This of course, would allow for a better grasp of the basic biology of these long established pathogens; those viruses which are now extinct but which may resurface, or even those viruses which are constantly in our minds as agents of natures bioterrorism. They conclude that “a long overdue renaissance in vaccinology has commenced and it is with anticipation and excitement that we wait to see progress in the next decade”.
Mahalingam S, Damon IK, & Lidbury BA (2004). 25 years since the era... Read more »
Mahalingam S, Damon IK, & Lidbury BA. (2004) 25 years since the eradication of smallpox: why poxvirus research is still relevant. Trends in immunology, 25(12), 636-9. PMID: 15530831
Mueller S, Coleman JR, Papamichail D, Ward CB, Nimnual A, Futcher B, Skiena S, & Wimmer E. (2010) Live attenuated influenza virus vaccines by computer-aided rational design. Nature biotechnology, 28(7), 723-6. PMID: 20543832
Racaniello VR, & Baltimore D. (1981) Molecular cloning of poliovirus cDNA and determination of the complete nucleotide sequence of the viral genome. Proceedings of the National Academy of Sciences of the United States of America, 78(8), 4887-91. PMID: 6272282
Wimmer E, Mueller S, Tumpey TM, & Taubenberger JK. (2009) Synthetic viruses: a new opportunity to understand and prevent viral disease. Nature biotechnology, 27(12), 1163-72. PMID: 20010599
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