Determinants of Enteric Calicivirus Infection.

PROJECT SUMMARYHuman norovirus (HuNoV) gastroenteritis is a significant public health burden worldwide. The lack of a robustand reproducible HuNoV cell culture system still limits our ability to study fundamental aspects of HuNoVinfection. While several recent breakthroughs increased our ability to propagate HuNoVs in vitro (e.g. B celland human enteroid cultures), these systems are not robust enough, can be time consuming and expensive orhard to replicate. The surrogate models available for HuNoV research do not necessarily reflect essentialbiological features of HuNoVs and their natural host. In this proposal we will use our novel rhesus entericcaliciviruses (ReCV) model that reflects the biological features and diversity of HuNoVs to identify hostdeterminants of infection. Zoonotic/interspecies transmission of ReCVs and HuNoVs between human and non-human primate hosts suggests evolutionary conservation of shared factors of host susceptibility between thetwo genera. Here we seek to identify host determinants of enteric calicivirus infection using CRISPR-Cas9genome wide screening. We recently identified a functional ReCV entry receptor that is necessary for ReCVpermissiveness in cell culture. We hypothesize that the ReCV entry receptor is also involved in HuNoVinfections. This hypothesis will be tested in the following specific aims. Aim 1. Characterize receptor mediatedReCV infection. Aim 2: Comparative characterization of HuNoV and ReCV infections in enteroid and B cellcultures. In the first aim we will dissect the role of HBGAs in ReCV receptor mediated entry by using ReCVisolates with disctinct HBGA binding, cell lines expressing different HBGAs and/or the receptor, EnterobacterSENG-6 EPS and different synthetic HBGAs. We will also evaluate the role of the different transmembraneisoforms of the receptor in infection and map the ReCV interaction site and importance of glycosylation. In thesecond aim, we will evaluate the role of ReCV entry receptor in HuNoV infections in enteroid and B cellcultures and identify other cell surface components playing a role in HuNoV infections. We will also evaluatethe mechanism of bile or bile salts in promoting HuNoV infections and the role of M cells in infections ofpolarized cells. Our long-term goal is to identify and characterize viral and host determinants of ReCV andHuNoV infections and to develop novel intervention/prevention strategies. Our proposal uses a novel entericcalicivirus model to understand viral entry and the role of bile and HBGAs in enteric viral infections. Ourfindings with ReCVs may be directly transferable to HuNoV infection. The major significance of this project isthe identification of determinants of susceptibility to enteric calicivirus infection that can lead to improvedHuNoV cell culture systems and new intervention/prevention strategies.