Fighting dengue with mosquito semen

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.

 Why dengue?

Mosquito sex

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

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

Viruses, vaccination and the inflammasome. Part 1.

The inflammasome

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.



Generic inflammasome
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.



IL-1beta.
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

Kanneganti, T. (2010). Central roles of NLRs and inflammasomes in viral infection Nature Reviews Immunology, 10 (10), 688-698 DOI: 10.1038/nri2851

Lamkanfi M (2011). Emerging inflammasome effector mechanisms. Nature reviews. Immunology, 11 (3), 213-20 PMID: 21350580

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

Virus of the week: MUMPS VIRUS (MuV)

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

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 = European communicable disease bulletin, 16 (8) PMID: 21371413

An ecological perspective on bat viruses

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. 

Abstract
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 are completely the opposite of what is seen with adenovirus where no changes in abundance were observed.

• And another where virus abundance did not significantly change over time – seen with the adenovirus - the complete opposite to the corona- and astroviruses. This may demonstrate basic differences in virus transmission, with adenoviruses persisting long-term in hosts and do not therefore rely on a readily susceptible population of hosts. These viruses have mechanisms of evading host immunity meaning that increases or decreases in protection do not lead to direct changes in adenovirus numbers.

This paper illustrates the potential importance that bat populations play in the ecology of viruses within general ecosystems shared with humans. This work, although not focussing on proven human pathogens, allows us to infer general principles of virus dynamics; these bat colonies may be able to signifcantly amplify RNA virus abundance across time, indicating potential periods of increased risk. The ability to accurately predict the chances of virus emergence may allow us to prevent future epidemics of seirous disease. But, the major questions to ask are whether this does accurately predict the behaviour of other viruses and whether even if virus numbers increase, will this even represent a signifcantly greater risk to other species? More work should be carried out to explore the role of not just bats but other animals in the spread of viruses through an entire ecosystem.


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) : 10.3201/eid1703.100526

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