Saturday, May 16, 2015

What is not known about Rabies infection

Greetings

Fig 1: Rabies Virus. Source
Rabies is a virus that needs no special introduction, both in terms of disease and fatality. It happened so, I was into a long discussion about what really makes Rabies so deadly. Though a straightforward answer seems to be "we don't really know", there seems to be a lot of theories that I could see from the literature.

Rabies is a member of Lyssavirus (Rhabdoviridae family). The genome consists of negative sense single stranded RNA, roughly 12Kbp in size. The entire structure is encapsulated into a lipid envelope with a characteristic bullet shape. The life cycle consists of entry by breach of skin. The virus is probably taken into the epithelial cells by Clathrin mediated endocytosis and subsequently reaches the muscles. It has been speculated that there probably is some rounds of replication in the muscles and then subsequently is concentrated in the neuromuscular junction. From there virus moves into the neurons and moves via the neurons to the Central Nervous system. 

Fig 2: Rabies p75NTR endosomal
pathway. Source
Until very recently, nothing much was known about the mechanism of rabies transportation or uptake in neurons. The speculation was that Rabies used acetylcholine receptor and moved via trans-synaptic transfer. However, a very convincing study by Perlson etal; showed that the virus uses p75NTR (p75 neurotrophin receptor), and moves in acidic compartments within neurons. The interesting point is that p75 alone cannot move as fast, but when attached to Rabies particle, it moves fast, really fast. This indicates that the virus also influences cellular machinery and kinetics of transport. Of course, p75 independent transport has been shown, but seems to be very slow and mayn't account for pathogenesis. As Perlson captures it in a single comment, "Rabies not only hijacks the nervous system’s machinery, it also manipulates that machinery to move faster. We have shown that rabies enters a neuron in the peripheral nervous system by binding to a nerve growth factor receptor, responsible for the health of neurons, called p75. The difference is that its transport is very fast, even faster than that of its endogenous ligand, the small molecules that travel regularly along the neuron and keep the neuron healthy.”

The retrogade axonal transport is of great interest since inhibiting this step can be really useful in terms of therapeutics. Of all the proteins produced by Rabies, G protein (glycoprotein; component of membrane spike), is essential for viral transport, as has been shown in mutation studies. Further the degree of neuroinvasion can be correlated with G protein sequences. It has been speculated that there are alternative receptors for G protein varieties, (formal proof is lacking) which may influence invasiveness. Current leading models suggest that virus moves intact inside the neurons.

Photo 1: Negri bodies. Source
What happens inside the neurons with Rabies infection? For a longtime, the only thing well known fact was the Negri bodies. Negri bodies are not found in all the infected neurons. They represent sites of active viral replication. In common terms they are called as cytoplasmic inclusion bodies. The contents of Negri bodies include a variety of rabies proteins such as L, P, and N involved in viral RNA synthesis, host proteins (especially TLR3, NEDD4 and Hsp70). This is guarded heavily by N and P proteins forming a cage like structure. This protects them from degradation. Proteomic experiments have shown that there is a lot happening inside the neurons with rabies infection and most of the molecules that seems to be associated has to do with neuronal functioning. By testing for rabies antigens in whole brain through fluorescence, it is clear that Rabies mainly infects thalamus, pons and medulla. Cerebellum and hippocampus, is largely unaffected. This also explains the effect of rabies on vital functions and ultimately fatality. This also correlates with brain imaging studies.

Before I get into the discussion of how rabies kills, I need to digress a little bit. Rabies is considered as a unequivocal fatal condition. Except for a very few case reports of people surviving rabies without immune treatment, it appears to be one hell of a pathogen. There is a thrust of pages online recommending Milwaukee Protocol (or also known as Wisconsin protocol), as a possible therapeutic for Rabies. The scientific community is highly skeptic of the procedure. The idea is that if you could slow down the viral axonal transport you could give enough time for immune system to take over. Immune system is able to clearly fight of the infection provided sufficient time for humoral response is given. The same is the idea in rabies vaccination post exposure. Of the 41 patients who received Milwaukee Protocol, only 6 have survived. There is no clear explanation of why. Further when there is a 100% protection with existing PEP (Post exposure Prophylaxis) why another method appears to work by chance. So the question to ask would be is Rabies really 100% fatal? Perhaps not though popular notion is that it is. For example, studies have shown Rabies neutralizing antibodies in otherwise healthy individuals though this is a small proportion. Further these may be cross reactive antibodies. These are people with risk of infection from vampire bats who are not effected by virus. The current view is that it is not 100% fatal and some may even survive without any treatment. There are other influencing factors. But mortality is indeed high.

Ignoring the statistics, let us ask the question how Rabies actually kills. In other words what is the pathogenesis.

Fig 3: Rabies virus immune-evasion mechanisms.
Source
In study models using tissue culture cells (such as Macoy cell line), a variety of cytopathic effects are seen. However cytopathology depends on the cell line used and the strain of virus used. The ability to induce apoptosis has been studied and correlated with G protein. Additionally the process seems to be delayed and hence some cells maynot ever show an effect. Paradoxically, ability to inhibit the apoptosis is the hallmark of Rabies infection. This can be explained by the late production of G protein and requirement of it to reach a threshold level to trigger apoptosis very late in the cell cycle.

There are different theories to explain Rabies- Neuronal dysregulation and Neuronal death. More recently the hint is that it is a combination of both, in other words neuronal dysregulation followed by Neuronal degeneration / death. An equally competing model is the excitotoxicity model. Perhaps the most worked on in recent years is the immunological model. According to Excitotoxicity model, the infection leads to neuroanl hyperactivity, cells to outstrip their energy supply and eventually die. 

The immunological hypothesis suggests that the virus in itself is not pathogenic but the immune response triggered due to the infection, causes sever inflammation and subsequent bystander neural damage. Remember I just told you that TLR3 is there inside the Negri bodies. It has been speculated that the TLR3 sequestration probably is an evasion strategy. Also there are other strategies by which Rabies can inhibit the immune response from triggering. See Fig 3. However, it is not clear despite drastic measures by the virus to counter act immune activation, what actually triggers heavy inflammation. Interesting enough fatal encephalitic rabies is not necessarily accompanied by substantial inflammation and there is a huge variability in inflammatory response in aniaml models. Adding to the complexity, neuronophagia can vary substantially and neuronal apoptosis does not seem to have an important role (Link) in Human rabies encephalitis.

The gist of this discussion is that finally we don't know what actually happens.

ResearchBlogging.org
Schnell MJ, McGettigan JP, Wirblich C, & Papaneri A (2010). The cell biology of rabies virus: using stealth to reach the brain. Nature reviews. Microbiology, 8 (1), 51-61 PMID: 19946287

Gluska S, Zahavi EE, Chein M, Gradus T, Bauer A, Finke S, & Perlson E (2014). Rabies Virus Hijacks and accelerates the p75NTR retrograde axonal transport machinery. PLoS pathogens, 10 (8) PMID: 25165859

Lahaye X, Vidy A, Pomier C, Obiang L, Harper F, Gaudin Y, & Blondel D (2009). Functional characterization of Negri bodies (NBs) in rabies virus-infected cells: Evidence that NBs are sites of viral transcription and replication. Journal of virology, 83 (16), 7948-58 PMID: 19494013

Dhingra V, Li X, Liu Y, & Fu ZF (2007). Proteomic profiling reveals that rabies virus infection results in differential expression of host proteins involved in ion homeostasis and synaptic physiology in the central nervous system. Journal of neurovirology, 13 (2), 107-17 PMID: 17505979

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Gilbert AT, Petersen BW, Recuenco S, Niezgoda M, Gómez J, Laguna-Torres VA, & Rupprecht C (2012). Evidence of rabies virus exposure among humans in the Peruvian Amazon. The American journal of tropical medicine and hygiene, 87 (2), 206-15 PMID: 22855749

Hemachudha T, Ugolini G, Wacharapluesadee S, Sungkarat W, Shuangshoti S, & Laothamatas J (2013). Human rabies: neuropathogenesis, diagnosis, and management. The Lancet. Neurology, 12 (5), 498-513 PMID: 23602163

Fooks AR, Banyard AC, Horton DL, Johnson N, McElhinney LM, & Jackson AC (2014). Current status of rabies and prospects for elimination. Lancet, 384 (9951), 1389-99 PMID: 24828901

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