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Studying viral infection at the whole-organism level

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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.

Zebrafish viral infection: In blue are cell nuclei, green endothelial cells and red viral proteins.

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

ResearchBlogging.orgLudwig, 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

Written by Connor

February 5, 2011 at 12:00 pm

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

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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.

GFP-virus infected cells

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.

Diagramtic 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.

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.

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’.

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.

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.

ResearchBlogging.orgLemon, 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

Written by Connor

February 1, 2011 at 11:06 am

A Mouse Model of XMRV Pathogenesis?

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Through studying viral pathogenesis we seek to understand mechanistically how viral infection and replication causes disease in a particular host. This of course will be subject to a number of complex variables involving both the host and the virus such as: dose; genotype; virus receptor distribution of host tissues;the ability of the virus to replicate in those infected cells and the hosts response to that infection.

Although it is an extremely complex system, knowledge of it may allow us to develop certain preventative strategies alongside new treatments and therapies. That is why being able to study viral pathogenesis is important and why some may welcome a recently published paper reporting initial data (possible cell tropism and host immune response) from a mouse model of infection with xenotropic murine leukemia-related virus (XMRV), a possible novel human pathogen (and a close relation of natural mouse retroviruses).

Mus musculus - relative of Mus pahari. Just incase you forgot what a mouse looked like.

Originally identified in a number of human prostate tumour samples, XMRV has since had a conflicting scientific history (covered much better elsewhere), with some studies showing a link between infection and chronic fatigue syndrome (CFS) and prostate cancer. Others since have failed to detect such a link. Despite this, knowledge of how this virus could potentially interact with the human body would of course be useful to acquire.

This understanding has been blocked somewhat by the lack of a small-animal model – a cheap, easier and more ethical alternative to non-human primate studies and of course easier to study than humans. XMRV just does not infect normal lab mice (Mus musculus) and thus pathogenesis in this host does not occur and if pathogenesis doesn’t occur, we can’t study pathogenesis. This species of mouse doesn’t express the receptor that XMRV utilizes to gain entry into cells. Sakuma et al have therefore used Mus pahari, a wild asian relative which does express the receptor for XMRV and thus may tell us something about XMRV pathogenesis.

The group showed that XMRV was able to successfully infect M. pahari cells in vitro and also was able to infect M. pahari following injection of the virus into whole-animals. Following infection, they were able to screen mouse cells and tissues (such as blood) for the telltale signs of XMRV infection over 12 weeks; XMRV being a retrovirus, integrates a DNA copy of its RNA genome into host cells and PCR detection of this integration may allow us to infer XMRV infection. They also investigated the possible role of infectious virus being present in the animal and the of XMRV replication on host cell functioning.

Detection of XMRV DNA in particular tissues may allow us to infer how infection proceeds within the host and how it causes disease. Viral sequences were detected in blood cells, heart, spleen, brain, testis and prostate tissues, although detection was highly variable between mice and a clear picture of infection didn’t really emerge over any of the time periods. The group focused on the effect of XMRV on lymphocytic cell functioning (possibly a good place to start giving its apparent involvement in CFS, an immunological disorder): CD4+ T helper cells and CD19+ B cells being targeted within the spleen. Over all, in some of the mice, increased total white-blood cell numbers was observed early in infection indicating a possible deregulation of lymphocyte development, although this was by no means common and only slight.

The role of both mouse adaptive and innate immunity was also assessed and interestingly, the mice generated a robust antibody response to XMRV antigens yet no long-term studies were involved to see how the virus adapted. Viral genome sequences found within blood and spleen tissues were sequenced and those from the spleen only, displayed predominant G-to-A hypermutation possibly indicative of intra-tissue restriction of viral replication. Host-mediated mutation of viral genomes will therefore more likely result in highly defective viral sequences and possibly prevent future viral infections; one example is that of APOBEC3-like enzymatic reactions.

Of course, these data are rather preliminary with optimisation of infection expected to come later, but at the minute, how does this relate to what happens in humans and will this model actually be on any use?

  • The development of a permissive small-mammal model of XMRV infection described here will certainly facilitate scientific investigation as it has done for many other viruses although it must be remembered that what happens in a mouse may not happen exactly that way in humans.
  • This study, however does appear to shed light on cell tropism of XMRV and possibly its transmission – blood-borne (although this was looked at really).
  • The possible deregulation of host lymphocyte development may play a role in the pathologies associated with it in humans. The highly variable pathological outcomes may be due to the relatively less homogenous gene pool of M. pahari rather than anything to do with the virus – although this may be occurring.
  • The fact that these mice developed strong antibody responses to this virus may allow the development of vaccine strategies.

But, of course, the importance and relevance of this work will all come down to whether it is or is not a true human pathogen yet we will certainly benefit from this work if it turns out that it is. Expect a lot more from this group if it is.

Sakuma T, Tonne JM, Squillace KA, Ohmine S, Thatava T, Peng KW, Barry MA, & Ikeda Y (2011). Early Events in Retrovirus XMRV Infection of the Wild-Derived Mouse Mus pahari. Journal of virology, 85 (3), 1205-13 PMID: 21084477

Sakuma T, Tonne JM, Squillace KA, Ohmine S, Thatava T, Peng KW, Barry MA, & Ikeda Y (2011). Early Events in Retrovirus XMRV Infection of the Wild-Derived Mouse Mus pahari. Journal of virology, 85 (3), 1205-13 PMID: 21084477

Written by Connor

January 26, 2011 at 8:46 pm