Drivers of Dengue Within-Host Dynamics and Virulence Evolution

Dengue is an important vector-borne virus that infects on the order of 400 million individuals per year. Infection with one of the virus's four serotypes (denoted DENV-1 to 4) may be silent, result in symptomatic dengue 'breakbone' fever, or develop into the more severe dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Extensive research has therefore focused on identifying factors that influence dengue infection outcomes. It has been well-documented through epidemiological studies that DHF is most likely to result from a secondary heterologous infection, and that individuals experiencing a DENV-2 or DENV-3 infection typically are more likely to present with more severe dengue disease than those individuals experiencing a DENV-1 or DENV-4 infection. However, a mechanistic understanding of how these risk factors affect disease outcomes, and further, how the virus's ability to evolve these mechanisms will affect disease severity patterns over time, is lacking. In the second chapter of my dissertation, I formulate mechanistic mathematical models of primary and secondary dengue infections that describe how the dengue virus interacts with the immune response and the results of this interaction on the risk of developing severe dengue disease. I show that only the innate immune response is needed to reproduce characteristic features of a primary infection whereas the adaptive immune response is needed to reproduce characteristic features of a secondary dengue infection. I then add to these models a quantitative measure of disease severity that assumes immunopathology, and analyze the effectiveness of virological indicators of disease severity. In the third chapter of my dissertation, I then statistically fit these mathematical models to viral load data of dengue patients to understand the mechanisms that drive variation in viral load. I specifically consider the roles that immune status, clinical disease manifestation, and serotype may play in explaining viral load variation observed across the patients. With this analysis, I show that there is statistical support for the theory of antibody dependent enhancement in the development of severe disease in secondary dengue infections and that there is statistical support for serotype-specific differences in viral infectivity rates, with infectivity rates of DENV-2 and DENV-3 exceeding those of DENV-1. In the fourth chapter of my dissertation, I integrate these within-host models with a vector-borne epidemiological model to understand the potential for virulence evolution in dengue. Critically, I show that dengue is expected to evolve towards intermediate virulence, and that the optimal virulence of the virus depends strongly on the number of serotypes that co-circulate. Together, these dissertation chapters show that dengue viral load dynamics provide insight into the within-host mechanisms driving differences in dengue disease patterns and that these mechanisms have important implications for dengue virulence evolution.

TRPV4 is necessary for trigeminal irritant pain and functions as a cellular formalin receptor.

Detection of external irritants by head nociceptor neurons has deep evolutionary roots. Irritant-induced aversive behavior is a popular pain model in laboratory animals. It is used widely in the formalin model, where formaldehyde is injected into the rodent paw, eliciting quantifiable nocifensive behavior that has a direct, tissue-injury-evoked phase, and a subsequent tonic phase caused by neural maladaptation. The formalin model has elucidated many antipain compounds and pain-modulating signaling pathways. We have adopted this model to trigeminally innervated territories in mice. In addition, we examined the involvement of TRPV4 channels in formalin-evoked trigeminal pain behavior because TRPV4 is abundantly expressed in trigeminal ganglion (TG) sensory neurons, and because we have recently defined TRPV4's role in response to airborne irritants and in a model for temporomandibular joint pain. We found TRPV4 to be important for trigeminal nocifensive behavior evoked by formalin whisker pad injections. This conclusion is supported by studies with Trpv4(-/-) mice and TRPV4-specific antagonists. Our results imply TRPV4 in MEK-ERK activation in TG sensory neurons. Furthermore, cellular studies in primary TG neurons and in heterologous TRPV4-expressing cells suggest that TRPV4 can be activated directly by formalin to gate Ca(2+). Using TRPA1-blocker and Trpa1(-/-) mice, we found that both TRP channels co-contribute to the formalin trigeminal pain response. These results imply TRPV4 as an important signaling molecule in irritation-evoked trigeminal pain. TRPV4-antagonistic therapies can therefore be envisioned as novel analgesics, possibly for specific targeting of trigeminal pain disorders, such as migraine, headaches, temporomandibular joint, facial, and dental pain, and irritation of trigeminally innervated surface epithelia.