Host Defense Evasion Mechanisms of Rabies Virus

Rabies Virus

Rabies virus (RABV) is a prototypical neurotropic virus and is often counted as one of the most deadly zoonotic diseases. Both wild and domestic animals can be afflicted with the disease and it is most commonly spread to humans via the saliva of an infected animal entering a bite wound or scratch. In Asia, most rabies infections are caused by rabid dogs. However in the Americas, and recently Australia and Western Europe, bat rabies has become a public health threat. Rabies is present on all continents except Antarctica, but the majority of rabies related deaths occur in Asia and Africa (WHO fact sheet 99).

RABV infections occur mainly in remote, rural communities where measures such as vaccinating dogs to prevent animal to human transmission have not occurred. Due to lack of resources and preventative measures, 95% of rabies related human deaths occur in Africa and Asia. Most at risk populations generally do not have the ability to access nor the money for the rabies vaccine or post-exposure prophylaxis, turning a rabies infection into a death sentence (WHO fact sheet 99). Most patients only survive for seven days after the onset of symptoms (Schnell et al 2009).

There are two main phylogroups of RABV which cause two distinct types of symptoms. Classical rabies, also known as furious rabies, causes 70% of animal and human infections while non-classical or paralytic rabies causes the remaining 30% of cases. Classical rabies is the more well-known strain and is characterized by symptoms such as hydrophobia, hallucinations, excited behavior, hyperactivity, and occasionally aerophobia. Paralytic rabies is less dramatic, as the patient’s muscles slowly become paralyzed with the paralysis radiating from the infection site. The patient eventually falls into a coma and dies. This non-classical form of rabies is often misdiagnosed, leading to an underrepresentation of the incidences of rabies (WHO and Schnell et al 2009). Both forms of rabies cause death through cardiorespiratory arrest (WHO fact sheet 99).

Great progress has been made in developing new rabies vaccines and preventative measures, but much of the molecular mechanisms of rabies virus remain a mystery. Improved genetic manipulation techniques that allow for direct manipulation of the rabies viral genome have given researchers a more detailed picture of rabies pathogenesis and a greater insight into virus-host cell interactions (Schnell et al 2009). Understanding viral-host cell interactions are key to understanding how rabies virus is able to evade the host’s immune response and make its way from the site of infection to the central nervous system and the brain. A better understanding of these mechanisms could help improve neuronal labeling and nuerotracer studies and treatments for other central nervous system diseases (Schnell et al 2009).

Host Cell Mediated T-Cell Apoptosis

Infiltrating T cells are the host’s method of controlling infections in the nervous system (NS). However, the T cell response in rabies victims is insufficient as it is inactivated by the virus (Lafon, 2011). Immunohistochemical studies of brain and spinal cord slices of rabies victims showed that it was leukocytes and not neurons that were undergoing cell death (cited in Lafon, 2011). Infiltrating T cells have a difficult time in the NS as several neuropeptides and nuerotransmitters downregulate T-cell activity (Niederkorn 2006). Using attenuated RABV, it has been observed that infected brains have upregulation of molecules such as somatostatin that are involved in limiting T-cell activity in the NS (Weihe et al 2008). However, it is important to note that attenuated and fatal strains of RABV act in different ways so whether the encephalitic RABV elicits this response is uncertain.

Studies done in vitro and in vivo have shown that RABV uses an evasive strategy similar to that of tumor cells by upregulating certain surface molecules such as B7-H1, FasL, and HLGA that trigger apoptosis in T-cells (Lafon et al 2005; Mergret et al 2007). It has been found that in mice lacking the functioning FasL ligand, there was a lower level of T-cell apoptosis in the NS compared to control mice. This reduction in T-cell apoptosis is possibly a contributing factor to the lower rates of RABV morbidity and mortality in these mice. Similar experiments revealed that B7-H1 was upregulated in RABV infected brains (Fig 3) and B7-H1 deficient mice had lower morbidity and mortality rates. Interestingly it has been found that the high expression rate in RABV-infected NS is due to the fact that both infected neurons and non-infected neuronal cells like astrocytes express B7-H1 (Fig 3). In Figure 3B, only the infected cells are stained green, however you can see the red B7-H1 expressed in the non-green cells around the neuron (Lafon et al, 2008).

RABV uses the immunosubversive molecules FasL and B7-H1 to protect against the host’s T-cells. Cells expressing FasL and B7-H1 trigger apoptosis pathways in T-cells expressing the Fas and PD-1 ligands upon binding. When the upregulation of these immunosubversive molecules is blocked, RABV virulence is drastically attenuated. Essentially, RABV hijacks systems already in place to create a more immunoevasive environment.

Prevention of Neuronal Apoptosis

While some neurotropic viruses such as West Nile virus or polio virus effectively kill motor neurons, RABV actively seeks to prevent neuronal degradation. RABV enters the NS through a neuromuscular junction or by passing through a synapse and utilizes the central nervous system as a transport system to the brain. Neuronal cell bodies, and possibly dendrites, are used for viral propagation and the virions travel in a retrograde direction towards the brain. Due to its dependence on neurons and the neuronal network, it would suggest that the virulence of RABV correlates to the survival of neurons (Lafon 2011). In fact, motor neurons of non-human primates infected with RABV showed no signs of degradation four days post infection (cited in Lafon 2011).

This prevention of apoptosis and axonal degradation is caused by the cytoplasmic form of the glycoprotein (G). In vitro RABV induced neuronal death is rare, however many laboratory created, attenuated strains induce neuronal apoptosis (Lafon 2011). The survival of the neuron and continued axonal growth depend on signaling cascades within the cell. PDZ domains are used in signal transferring and assemble and or regulate certain protein networks (Prehaud et al 2010, Lafon et al 2011). The RAVB G protein contains a PDZ-binding cite (PDZ-BS) in its C terminus. The only difference between two laboratory RABV strains, VIR and ATT, is in their G protein sequence. However, VIR is more virulent strain causing fatal encephalitis in mice and ATT is an attenuated strain. It has been suggested that the Gln to Glu mutation in the C terminus PDZ-BS in the ATT G protein is what causes the attenuated phenotype by inducing neuronal apoptosis. In vitro, VIR was able to prevent nueronal apoptosis and maintain axon and dendritic growth while ATT was not (Fig 4). Instead, ATT actually triggered apoptosis in infected cells (Fig4) (Prehaud et al 2010).

The different phenotypes stem from the different binding specificities of the two PDZ-BS. The G protein of the virulent strain only interacts with microtubule associated serine-threonine kinase 1 and 2 (MAST1 and MAST2 respectively) while the mutated G protein interacts with three unrelated PDZ binding partners, the most interesting of which is non-receptor protein tyrosine phosphate 4 (PTPN4). PTPN4 silencing has been suggested to trigger apoptosis, and it has been demonstrated that ATT G protein silences PTPN4. However, un-mutated G protein binding to MAST2 causes nuerite outgrowth (Prehaud et al 2010). The specificity of the G protein suggests that RABV walks a fine line de-activating some signaling cascades while leaving others intact.

Neuroinflammation and the Blood Brain Barrier

The NS intrinsically limits the inflammation response following injury; however inflammation is still triggered in the NS by most infections. In contrast to most encephalitic viruses, RABV triggers a more limited inflammation response. In fact the more pathogenic the strain is, the smaller the inflammatory response (Baloul and Lafon 2003). It is unclear how exactly RABV is able to limit the inflammatory response, but it has been suggested that the ability correlates to the differences between classical and non-classical strains. By comparing the amount of viral RNA and 18 cytokine mRNAs in twelve different brain regions of dogs infected with classical and non-classical rabies, Laothamatas et al (2008) was able to determine key differences in the inflammatory response triggered. The differences were found early on in infection, with non-classical RABV infected dogs having higher levels of interleukin-1beta and interferon-gamma and lower levels of viral RNA. Dogs infected with classical RABV had much higher levels of viral mRNA in brain tissue. Later in infection, there wasn’t much difference between the viral RNA and cytokine mRNA levels in dogs infected with the different strains (Laothamatas et al 2008). The increased nueroinvasiveness of the classical strain probably correlates to the decreased immune response in comparison with the non-classical strain. Strength of the nueroinflammitory response might also correlate to the permeability of the blood brain barrier (BBB).

The permeability of the BBB in rabies infection is important as permeability relates to host cell survival. A more permeable membrane leads to an increased chance of host survival as it allows for passage of more immune cells into the NS. It has been shown that BBB permeability is increased in laboratory attenuated strains of RABV but until Chai et al (2014) the underlying mechanisms were a mystery. By comparing expression of tight junction proteins in the brain microvascular tissue of mice infected with either wild type or lab attenuated RABV, Chai et al (2014) were able to determine that the enhancement of BBB permeability was associated more with the differences in chemokine/cytokine expression rather than the virus itself. Figure 4 shows the network of inflammatory chemokines and cytokines triggered by each strain. The network induced by attenuated strain (Fig4 A) contains 26 focus molecules while the wild type strain network (Fig 4B) only has 12. IFN-gamma is at the center of the network, which corresponds to the findings of Laothamatas et al (2008) and their non-classical strain induction of INF-gamma. Upon further investigation of INF-gamma’s role in BBB permiability, Chai et al. (2014) found that INF-gamma silencing causes a decrease in TJ and an increased permeability of the BBB.

Conclusion

Overall paper length should be 2,000 (Draft 1), 3,000 words (Final), with at least 3 figures.

References

[Sample reference] Takai, K., Sugai, A., Itoh, T., and Horikoshi, K. "Palaeococcus ferrophilus gen. nov., sp. nov., a barophilic, hyperthermophilic archaeon from a deep-sea hydrothermal vent chimney". International Journal of Systematic and Evolutionary Microbiology. 2000. Volume 50. p. 489-500.

Edited by Alexandra Gonzales, student of Joan Slonczewski for BIOL 375 Microbiology, 2014, Kenyon College.