So, how do you know when a vaccine is safe?

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

Lewis 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 lewis 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: 10.1016/j.vaccine.2011.02.005

The ‘interactome’ of a host/pathogen triad

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

“The virus is ‘dead’. Long ‘live’ the virus.” – what does history say about how virology should develop in the future?

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 Mühlberger 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 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

Can we visualise virus infection as it happens - in real-time?

One worthwhile way to study viruses – and other micro-organisms – is to see where exactly they are found within a host. How do they enter the body? What organs do they infect and how? How do they spread from tissue to tissue and organ to organ? How do they exit the body? These are just some of the questions which it would be good to actually SEE how and where it happens. Maybe then we could better understand the dynamic relationships governing infection and disease; maybe then we could design better, more effective therapies. Just maybe. Like it is all that easy.

How could we track adenovirus movements?

For one thing viruses are pretty small - so, are there any high resolution methods of looking at infection that may be able to help us? Pathologists can look at tissue samples from living or dead patients (animals included) and microscopically assess these for signs of viruses using for example: antibodies specific for particular proteins or nucleic acid probes (in situ hybridisation or PCR perhaps) found only in infected cells. This stuff is pretty good and is routinely used for both, clinical sciences and biological research but its kind of limited and in the last decade or so, the use of ‘reporter genes’ - like GFP and luciferase has facilitated the easier and faster analysis of viral infection.

Reporter genes are inserted into the viral genome and when expressed upon cell infection, they produce a protein – maybe a fluorescent or luminescent protein – whose function can be assayed and followed. This is what we can look at during infection. There are however certain limitations with this, in that, only those viruses that successfully infect a cell will express the reporter gene; viruses which fail to infect and are taken up by the liver or immune cells will not be detected. We are seeing a biased image of viral infection if we only consider reporter gene expression. This is particularly important when we are using viruses as therapeutic agents themselves as strict pharmacological testing requires intimate details of in vivo distribution and kinetics. So how can we see viruses in vivo without reporter genes?

As a paper in PLoS ONE, from a group at the Mayo Clinic in Rochester, demonstrates, there IS an alternative way to track viruses in vivo – the molecular attachment of individual reporter molecules to the virus itself thus requiring no gene expression and therefore allowing a more unbiased view of infection. An interesting aspect of this work is that it is carried out in real-time; these reporter molecules can be visualised as infection happens, at the millisecond scale. This also allows for the tracking at very early time points, times where no viral gene expression is taking place.

This study came at it from the angle of developing safer and more effective anticancer viruses, viruses which will infect and kill only those malignant cancer cells but this could be applied to any area of investigation. Being able to follow virus distribution in a mouse-model is of course a great scientific and clinical benefit to them. The group dyed an adenovirus vector with a molecule which emits light in the near-infrared range (particularly suited to in vivo imaging) which they then injected into groups of mice via their jugular vein. They were thus able to analyse and quantify the tissue distribution of their labelled vector throughout a whole mouse, in real-time.

This is the first time that this kind of imaging has been carried out and it certainly won’t be the last. This work could be applied to yet more viral vectors; it could be used to study a ‘natural’ infection or it could be used for non-viral imaging of therapeutic nanoparticles. A combination of this early time-point analysis with later, reporter gene expression imaging would be able to give us an unprecedented view into dynamic real-time viral infections in a number of model systems.

Brandenburg, B., & Zhuang, X. (2007). Virus trafficking – learning from single-virus tracking Nature Reviews Microbiology, 5 (3), 197-208 DOI: 10.1038/nrmicro1615

Hofherr, S., Adams, K., Chen, C., May, S., Weaver, E., & Barry, M. (2011). Real-Time Dynamic Imaging of Virus Distribution In Vivo PLoS ONE, 6 (2) DOI: 10.1371/journal.pone.0017076

The Grand Challenge of Aerosolised Vaccines

Despite the development of effective vaccines, many human populations are currently at the mercy of numerous endemic viral pathogens. Measles virus is one such pathogen that, in 2008, was responsible for 164,000 deaths; the worst effected areas are South-East Asia and Africa (WHO stats can be found here). You might find this surprising as there is currently a very good measles vaccine in use – in fact you probably received at some point during childhood and are protected from future infection. Measles cases have been significantly reduced in the developed world, so why hasn’t this vaccine allowed for the eradication of measles virus transmission in the developing world?

Needle vaccination against measles

The key to controlling measles – and other viruses – is to generate sustained high levels of good quality immunity within a population so that the virus can no longer successfully infect and has nowhere to go; this is known as herd-immunity. The problem then is, well why can’t we achieve the herd immunity required to prevent virus transmission? In places like Africa, where people are reminded daily of the horror of measles, you don’t have to force them to accept vaccination unlike what was seen in the UK and US recently so they are readily vaccinated. One problem however, appears to be the mode of vaccination, that is injecting the virus vaccine creates hurdles to a successful immunisation campaign:

·      Trained healthcare workers are required to safely administer the vaccine when it is injected

·      The currently used vaccine formulation tends to go off in temperatures ~ 37 degrees Celsius causing problems for transport and storage especially in areas such as Africa.

·      The use of used/contaminated needles may facilitate the problems of blood-borne diseases and drug use

Are there any alternatives to needle vaccination?

There are of course other ways to vaccinate people, maybe the respiratory tract or the gastrointestinal tract may make better options – especially considering how different injecting a virus is to most of their natural entry mechanism. The mucosa-associated lymphoid tissue, lining the mucosal epithelium of thegastrointestinal and respiratory tracts may also prove to be a more effective place to induce stronger immune responses.

Currently, some 3million children have already been successfully vaccinated from measles using a ‘wet’ aerosol delivery system, however the formulation was unstable above 4 degrees Celsius and delivery was difficult. A recent paper published in PNAS has sought to improve upon this current measles vaccination technology (and also get around the problems of injecting vaccines) through the generation of a highly immunogenic respiratory-delivered ‘dry’ vaccine formulation. It was tested in macaques, can be administered as a single dose and is a highly thermostable, powdered formulation.

'PuffHaler' aerosol delivery system for 'dry' vaccines

 So, how good is it?

They report that their aerosol-delivered vaccines were deposited into the upper and lower respiratory tracts and resulted in the generation of good-quality measles virus specific humoral (B cell + antibody) and cellular (CD8+/CD4+ T cells) immune responses without safety concerns; there also exists a long-lived (<1 year) B cell memory function (and some T cell memory) correlating with long-term virus protection. They show the their vaccine strategy allows for the successful protection from subsequent measles virus challenge. Through comparisons with injected vaccine, the group were able to show that indeed all routes of vaccination generated the required level and quality of immunity required to protect from measles but given other concerns with injecting vaccine aerosol delivery may prove better. These results indicate that this proof of concept, novel vaccine may be comparable to the previously used formulation – although human studies would have to be carried out.

Aersoal delivery system

Scientists are being allowed to investigate problems like these only through being funded by the 'Grand Challenge in Global Health Grants' via the Bill and Melinda Gates Foundation; the money supplied allows for the development of better, safer and ultimatey more cost-effective vaccines. This work highlights the importance in the development of these new and more effective vaccine technologies in order to facilitate the eradication of viral pathogens worldwide. How might this method of administration affect other vaccines, only further work will decide but if we are unable to prevent measles transmission with a highly effective vaccine then what hope do we have to prevent other, less well-studied viruses without decent vaccines?

Lin, W., Griffin, D., Rota, P., Papania, M., Cape, S., Bennett, D., Quinn, B., Sievers, R., Shermer, C., Powell, K., Adams, R., Godin, S., & Winston, S. (2011). Successful respiratory immunization with dry powder live-attenuated measles virus vaccine in rhesus macaques Proceedings of the National Academy of Sciences DOI: 10.1073/pnas.1017334108

HIV & Measles - double hit pathogenesis?

Despite ongoing worldwide eradication efforts, measles infection still results in significant morbidity and mortality. Although, throughout most of the developed world measles infection has been considerably reduced there still exists massive (and deadly) outbreaks in areas such as Africa and South-East Asia. Investigation of the reasons why this disparity occurs therefore  is of major medical, political and social interest.

Many factors are likely to be behind this major difference - and all of which deserve our attention if we are ever to remove measles from the human population. There exists problems in rolling out vaccines in countries with poor infrastructure such as roads and transport facilities; disruption to what is known as the vaccine 'cold-chain' (vaccines have to be kept cold to avoid rendering them unusable) is likely to occur; general poor health of the population in these regions and possible interference of vaccination in children with high levels of passively acquired maternal antibody.

Measles vaccination efforts in Africa may not be entirely effective

Today in PLoS Pathogens, Nilsson and Chiodi highlight in a featured opinion article, another possible source: the link between co-infection with HIV-1 and Measles infection. They point out that HIV-1 infection and replication may result in impaired immune responses in both mothers and children leaving open the possibility of measles infection (no immune system, no protection). HIV-1, as I'm sure you will all know, is a potentially deadly pandemic retrovirus - particularly a major problem in sub-Saharan Africa- which infects humans where it resides in the bodies own immune system: T cells, dendritic cells and macrophages. Viral replication results in the death of these immune cells and destruction of important lymphoid tissues resulting in an individual without key immune functions.

The authors note that children born to mothers who are HIV-1 positive or are HIV-1 positive themselves develop lower levels of anti-measles antibody upon vaccination -a big deal if we're looking to protect these kids through vaccination. They show that memory B cells may be impaired and lower protection will result through failure to mount a B cell-generated antibody response. Immunity is a highly regulated system, if you remove one aspect-  in this case T cells - you will affect another pathway , in this case B cells. Thus there exists a major  problem with HIV-1 infected people and infection with other pathogens in the environment; HIV-1 infection significantly alters the host immune system weakening it to other invading pathogens such as measles which is endemic in these areas.

So how do we overcome this problem? Well, the authors suggest that on top of increasing vaccination coverage through catch-up programs it would be wise to administer anti-retroviral drugs  to mothers and children prior to vaccination to allow sufficient immune function; this should hopefully make a difference in combating both measles and HIV in the developing world, especially in an area where both cause so much pain. Hopefully, strategies such as this will aid treatment efforts for other pathogens rife in the developing world - targeting both HIV and the individual agents may be more effective.

Sadly, there exists another interaction between HIV and co-infection with other pathogens. Infection usually results in increased levels of immune cells in the blood and tissues yet these very cells are the target for HIV and if these cells increase, HIV replication will also. There exists a deadly interaction between multiple pathogens which must be broken.

Nilsson, A., & Chiodi, F. (2011). Measles Outbreak in Africa—Is There a Link to the HIV-1 Epidemic? PLoS Pathogens, 7 (2) DOI: 10.1371/journal.ppat.1001241

The molecular domestication of amphibian retroviruses - do they play aphysiological role?

Xenopus tropicalis - do recently identified ERVs play a functional role?

We mostly think of viruses of being ‘bad’ and ‘dangerous’ yet there are countless examples of viruses playing a positive role in their host’s life. These symbiotic agents have been co-opted by the host to do something good; some viruses have even been inserted into our genomes and thus are forever tied to our germline and our descendants - sometimes even these viruses can do good. This is the kind of game evolution plays with our viral parasites and us – its generally pretty cruel and inconsiderate but every so often we get something good out of it.

One example of these endogenous viruses is the endogenous retroviruses or ERVs, which are somewhat related to the non-endogenous – ‘exogenous’ – retroviruses that can cause disease in us and other animals (HIV XMRV?). Just to put it into perspective, 8% of our genome is made up of these ERVs and they also make up a large chunk of other vertebrate genomes. The majority of these inserted retroviral genomes have been destroyed by the forces of evolution and thus bear little resemblance to their ‘wild’ cousins; they are not expressed and their sequence shows little homology to other retroviruses. However, some ERVs have changed very little, suggesting an important function may be conserving them – these are expressed and do sort of resemble the exogenous ones. The insertion of a novel ERV sequence into a host’s genome acts as evolutionary raw material allowing significant adaptive functions to arise and a great deal of evidence suggests they can these can even play a physiological role in host biology – this is known as molecular domestication. One interesting example is the ERV role in the mammalian placenta.

A recent paper reports the discovery and characterisation of an amphibian ERV whose genomic organisation is highly conserved making it a good candidate to have a novel physiological function. Investigating the genome of Xenopus tropicalis - an 'African clawed frog', the group discovered a unique DNA sequence that was highly related to a previously characterised Xenopus protein with frost-resistant functions - allowing winter survival in woodland frogs. This 9,551 base-pair DNA sequence not only contained the intact frost-resistant gene but also a full-length retroviral genome with the general organisation of many common ERVs – 5’ LTR-GAG-POL-ENV-3’LTR.

ABSTRACT: We report on the identification and characterization of XTERV1, a full-length endogenous retrovirus (ERV) within the genome of the western clawed frog (Xenopus tropicalis). XTERV1 contains all the basic genetic elements common to ERVs, including the classical 5'-long terminal repeat (LTR)-gag-pol-env-3'-LTR archi- tecture, as well as conserved functional motifs inherent to each retroviral protein. Using phylogenetic analysis, we show that XTERV1 is related to the Epsilonretrovirus genus. The X. tropicalis genome harbors a single full-length copy with intact gag and pol open reading frames that localizes to the centromeric region of chromosome 5. About 10 full-length defective copies of XTERV1 are found interspersed in the genome, and 2 of them could be assigned to chromosomes 1 and 3. We find that XTERV1 genes are zygotically transcribed in a regulated spatiotemporal manner during frog development, including metamorphosis. Moreover, XTERV1 transcription is upregulated under certain cellular stress conditions, including cytotoxic and metabolic stresses. Interestingly, XTERV1 Env is found to be homologous to FR47, a protein upregulated following cold exposure in the freeze-tolerant wood frog (Rana sylvatica). In addition, we find that R. sylvatica FR47 mRNA originated from a retroviral element. We discuss the potential role(s) of ERVs in physiological processes in vertebrates.

Following the characterisation of the genome sequence, the group looked whether there any more ERVs like this one in Xenopus genomes  to see if  this a rare example of a highly conserved ERV and were there any other examples of these sequences present? There turned out to be 59 genomic loci with some sort of homology to the newly found ERV however all had significant mutations present rendering them functionally inactive – at least where gene expression is concerned. These sequences were mapped onto Xenopus chromosomes, showing that the intact ERV was present on chromosome and the ‘damaged’ ones were found throughout the genome. This ERV is after all a lone agent in the Xenopus genome - confirmed by these experiments.

Phylogenetic studies were also carried out which suggested that primary retroviral integration occurred roughly 41 million years ago and from then on multiple rounds of movement around the genome or reinfection generated the many mutated copies around the genome. Their results also suggest that this ERV is actively replicating and inserting itself into the genome up to the present day. A cousin of this retrovirus was also found in the closely related X.laevis genome showing that integration occurred prior to the evolutionary separation of these two lineages.



They next turned their attention to whether this ERV had a functionally active role (is it transcribed; in what tissues and at what points in frog development?) in host biology as observed in other host/ERVs. Using real-time PCR and in situ hybridisaton techniques, the group were able to follow ERV expression throughout X. tropicalis development and assess the level of transcription and tissue localisation and possible infer a physiological function. They noted a highly regulated yet dynamic expression of gag, pol and env expression from fertilisation through metamorphosis (curiously a peak of activity was seen during metamorphosis) and adult life but does this control of expression actually mean something functional or is it merely physiological neutral? This ERV may just be replicating within the host genome without contributing something to host life. In order to understand this, they subjected X. tropicalis tadpoles or cell lines to a number of biological ‘stresses’ e.g. metabolic, temperature and UV stresses. An upregulation of ERV expression was seen upon metabolic and UV stresses and not in temperature – suggesting a fine tuning of its expression in response to a number of stresses. Whether this actually achieved something functionally was not investigated.



A recently discovered retrovirus derived gene in another frog species was found to play a role in protecting frog cells from the effects of freezing conditions. This study, on the backs of that investigation determined that frost-tolerant gene was derived from a highly conserved ERV present within Xenopus genomes. A distinct physiological role for these ERV-derived genes was not validated in this study yet in the future, further characterisation of its expression in vivo under temperature stress should be undertaken. This work underlines the importance that retroviruses and their endogenised cousins play in host cell functioning and evolution. Viruses are not all bad news – sometimes they can help you.

Roossinck, M. (2011). The good viruses: viral mutualistic symbioses Nature Reviews Microbiology, 9 (2), 99-108 DOI: 10.1038/nrmicro2491

Sinzelle, L., Carradec, Q., Paillard, E., Bronchain, O., & Pollet, N. (2010). Characterization of a Xenopus tropicalis Endogenous Retrovirus with Developmental and Stress-Dependent Expression Journal of Virology, 85 (5), 2167-2179 DOI: 10.1128/JVI.01979-10

Studying viral infection at the whole-organism level

Some questions about how viruses cause disease in their hosts (viral pathogenesis) are best asked and studied using an in vivo model system; just sometimes infecting cells under tissue culture conditions just doesn't cut it. Questions like: how does a virus interact with all the immune cells during an infection and what cells does the virus actually infect should be asked this way.

But of course, this in vivo stuff is a great deal more difficult than in vitro studies and appropriate animal models don't just grow on trees; this is why, when a relevant model system of viral infection comes along we get excited - well at least I get excited. Unless you look at everything in its entirety, you never know what you will miss and viruses being as small as they are, its easy to miss something important and missing something important is bad news in the world of science.



A recently published study has looked at viral infection at the 'global' or whole-organism level using transgenic zebrafish larva infected with Infectious Hematopoietic Necrosis Virus (IHNV), an RNA virus related to rabies virus and is particularly deadly if you happen to some form of salmonid. Zebrafish are generally pretty good models for a whole lot of biological processes: zebrafish genetics are pretty well understood allowing for easy transgenics; they are particulary easy to study, especially to image as they are small and transparant and some genes/pathways are well conserved with humans meaning that it may have some applications to us. These factors all suggest that zebrafish may be a pretty decent model to understand viral infection in general, not just in fish.

Following infection, they were able to look at entire whole organisms for viral presence, concentrating on what particular cells/organs contain viral mRNA  and proteins. They were able to follow infection through its entirety, at early stages and the later stages when serious disease takes hold, allowing the elucidation of intra-host viral spread and dissemination. They used their system to shed light on the mechanisms of IHNV pathogenesis, showing that viral infection led to vascular endothelium destruction and impaired blood flow. It is just near impossible or at least a lot of hard work to do this kind of analysis in any other model system.

[caption id="attachment_161" align="aligncenter" width="300" caption="Zebrafish viral infection: In blue are cell nuclei, green endothelial cells and red viral proteins."][/caption]

Using this model - as in all model systems - comes with certain caveats attached: IHNV is not a natural pathogen of zebrafish (indeed, to date no viruses have been described) , i.e. what we see here may not be exactly what happens out there in the real world when this virus infects salmon. The virus was also injected into the bloodstream of these fish which is highly unlikely to occur in the wild - how would the infection change if it were administered another way? Not considering these issues, this work offers up a decent picture of systemic dissemination of IHNV in a not-so-perfectly matched host. Only time will tell how applicable to the real-world this is.

Its hard to imagine this work being carried out in any other kind of vertebrate - transparent rats in the future perhaps? But this stuff has been carried out using GFP expressing viruses within a non-human primate model only a week before this. Although not as easy to image, hard-work and dedicatedly searching through cells and tissues for signs of infection allows us to understand viral infection at the whole-organism level more appropriate to human disease.

This pretty much makes their statement below a bit incorrect, or at least out-dated:

We describe in this paper the spread of a viral infection throughout an entire organism, something that, to our knowledge, has not been done before in a vertebrate.

As a final thought, wouldn't it be great to image viral infection in real-time using a GFP-expressing IHNV in this zebrafish model? - just checked, there is a GFP IHNV virus out there. Check out the live-cell imaging of GFP neutrophils in a zebrafish above.

Two Zebrafish larvae

Ludwig, M., Palha, N., Torhy, C., Briolat, V., Colucci-Guyon, E., Brémont, M., Herbomel, P., Boudinot, P., & Levraud, J. (2011). Whole-Body Analysis of a Viral Infection: Vascular Endothelium is a Primary Target of Infectious Hematopoietic Necrosis Virus in Zebrafish Larvae PLoS Pathogens, 7 (2) DOI: 10.1371/journal.ppat.1001269

Ludwig, M., Palha, N., Torhy, C., Briolat, V., Colucci-Guyon, E., Brémont, M., Herbomel, P., Boudinot, P., & Levraud, J. (2011). Whole-Body Analysis of a Viral Infection: Vascular Endothelium is a Primary Target of Infectious Hematopoietic Necrosis Virus in Zebrafish Larvae PLoS Pathogens, 7 (2) DOI: 10.1371/journal.ppat.1001269

Can fluorescent-‘labelled’ viruses illuminate their mechanisms of pathogenesis?

Have you ever wanted to visualise viral infection? Ever wanted to observe how they enter and spread throughout their host organism? Ever wanted to know how exactly they caused disease - at the cellular and whole-organism level? Well, this may be entirely possible using fluorescent-labeled recombinant viruses infecting a relevant model system.

[caption id="" align="aligncenter" width="504" caption="GFP-virus infected cells"][/caption]

So how does it work?

Lemon et al recently report the continued investigation of measles virus pathogenesis in a non-human primate (Macaque) model utilising a green-fluorescent protein (GFP) expressing virus. Upon infection of host cells, viral transcription leads to the very high expression of GFP, flooding the cytoplasm with this fluorescent ‘tag’. Subsequent microscopy, imaging and immunohistochemistry allows for the identification and location of the infected cells, tissues and organs - see image above. Tracking of cellular infection allows us to decipher the development of MeV entry, spread and replication at both the cellular and whole-organism level throughout the entire infection. Studies such as these give an unprecedented view of viral infection in a means directed related to that of human infection. This model even allows for macroscopic real-time detection of fluorescence and hence viral infection.

Why is this important for measles?

Despite a highly effective vaccine and significant global control initiatives, measles infection still accounts for significant morbidity and mortality worldwide, mostly in the developing world (164,00 deaths in 2008). This is mostly attributable to the profound immunosuppression induced allowing for further infection with opportunistic pathogens. Currently, much is known about measles pathogenesis yet the molecular mechanisms of such are poorly understood and it is therefore of great interest to better understand these processes by which MeV infects and causes disease in humans. Knowledge of such may facilitate the development of more effective and safer vaccines for measles and indeed other viral pathogens.

Viruses being obligate intra-cellular parasites, must enter and exit cells in order to survive. Most of viral pathogenesis can therefore be attributed to the effects of viral replication of host cells and tissues; a major determinant of which is the expression of receptors on host cells surfaces allowing viral entry, infection and replication. Currently only a single receptor – CD150 - (otherwise known as signalling lymphocyte activation molecule SLAM) has been discovered that wild-type pathogenic MeV uses to enter host cells; the distribution of which only explains part of measles pathogenesis as epithelial and neuronal cells (important target cells) do not express the protein. As indicated by this receptor being expressed on lymphocytes and other immune cells, MeV is a highly lymphotropic virus! But if epithelial cells fail to express the receptor on their surface, how come its possible for MeV to enter via these cells?

The classical view of measles pathogenesis was that free-virus entered the host through the respiratory route, infecting and primarily replicating within the epithelial cell lining of the respiratory tract. Newly produced virus spreads to nearby lymph nodes where infected monocytes – a type of immune cell - facilitates viral dissemination throughout the host, resulting in the well-known symptoms of measles. The problem with this being that epithelial cells and unstimulated monocytes fail to express the MeV receptor CD150 and infection should therefore not occur. Recently, it has been shown (again using a GFP expressing virus in a macaque model) that MeV predominately infects dendritic cells during the peak of infection, ruling out a major role for monocytes. There is also however no direct evidence of MeV primary replication within the epithelium of the respiratory tract at the early stages of infection. So what exactly happens during the start of infection and does it develop? GFP-expressing viruses may shed light on this question.

[caption id="" align="aligncenter" width="415" caption="Diagramatic representation of the cellular composition of the human respiratory tract - notice the epithelial cell lining and the alveolar macrophages. Dendritic cells are however not shown on this diagram."][/caption]

So how can we study the early stages of infection?

The incubation period of  measles is about 2 weeks in humans making it particularly difficult to study the early events of viral infection – the kind of events like host entry, initial site of replication and subsequent intra-host dissemination - this is where we can use a non-human primate model.

Lemon et al  generated a highly virulent recombinant MeV based on viral isolates from an outbreak in Sudan; they engineered the viral genome so that it expressed GFP upon entry into cells – an addition that causes little or no replication defects to the virus. Groups of macaques were subsequently infected via the respiratory route allowing highly sensitive visualisation of GFP expressing cells following necropsy. The early time-points of around 5 days post infection were focussed on in this investigation allowing the determination of the early cell targets - epithelium? Immune cells?

So what did they find?

Their results suggest that at the early stages of MeV infection, GFP and hence viral replication is only found in immune cells within the respiratory tract and not the epithelial lining. Dendritic cells and alveolar macrophages are believed to capture viral particles in the lungs allowing spread via infected cells. This is known as a Trojan horse entry mechanism like that used by HIV to pass through mucosal tissues and infect humans - see below. This infection allows for spread and localised replication within nearby lymphoid tissues and then on to draining lymph nodes where massive lymphocyte cell infection may occur facilitating dissemination throughout the host, mainly within lymphoid tissues. Virus can be carried through host blood vessels to other lymphoid target tissues like the tonsils and adenoids and the gut-associated lymphoid tissue ' Peyer's patches'.

[caption id="" align="aligncenter" width="465" caption="HIV entry mechanisms utilising dendritic cells to pass through epithelial cell barriers - the 'Trojan horse' mechanism. This may be directly analogous to MeV entry and primary spread except in the respiratory tract."][/caption]

What does this mean?

This study clearly demonstrates the importance of non-epithelial cells such as dendritic cells in MeV entry, early replication and subsequent systemic spread. It does not however, rule out a major role for epithelial cells in later stages and in transmission - MeV still infects non-CD150 expressing cells and currently the mechanisms of which are unknown. Focusing on the later stages of infection may allow us to appreciate the other cell targets in pathogenesis and viral transmission. As mentioned previously, the use of fluorescent-labeled viruses offers an unprecedented view of viral entry, spread and pathogenic mechanisms. We should look forward to the time when studies like these are applied to other viral and indeed non-viral pathogens.

Lemon, K., de Vries, R., Mesman, A., McQuaid, S., van Amerongen, G., Yüksel, S., Ludlow, M., Rennick, L., Kuiken, T., Rima, B., Geijtenbeek, T., Osterhaus, A., Duprex, W., & de Swart, R. (2011). Early Target Cells of Measles Virus after Aerosol Infection of Non-Human Primates PLoS Pathogens, 7 (1) DOI: 10.1371/journal.ppat.1001263

Coombes, J., & Robey, E. (2010). Dynamic imaging of host–pathogen interactions in vivo Nature Reviews Immunology, 10 (5), 353-364 DOI: 10.1038/nri2746