Hantaviruses cause hantavirus cardiopulmonary syndrome (HCPS) and hemorrhagic fever with renal syndrome (HFRS) with case fatality rates of up to 40% and 14.5%, respectively. FDA- approved hantavirus drugs and vaccine do not exist. Recent advances in RNA sequencing technology has led to the discovery of more than 40 genetically distinct hantaviruses carried by rodents, insectivores (moles and shrews) and bats. Consequently, chances of zoonotic spillover are likely to be higher in future as deforestation, habitat destruction and climate change will bring more of the hantavirus reservoir hosts in closer proximity to the human populations. Despite their huge genetic diversity and public health importance, our understanding of the molecular determinants of hantavirus susceptibility (capacity for virus entry) and permissivity (capacity for virus replication) at the cellular level is derived from studies of only a few hantaviruses and remains rudimentary. This is largely due to the lack of molecular tools and hantavirus isolates and the general need to study hantaviruses in biosafety level-3 (BSL3) containment. Surrogate viruses such as recombinant vesicular stomatitis viruses (rVSVs) pseudotyped with the hantavirus entry Gn/Gc glycoproteins provide excellent tools for investigating virus entry and virus-host interactions under BSL-2 containment. Given that the N-terminal domain of Gn (Gn- NTD) forms most of the surface-exposed part of the Gn/Gc spikes on the virion surface and is the most divergent region of Gn/Gc, we hypothesize that (i) Gn-NTD diversity is a key viral determinant of hantavirus susceptibility, and (ii) our recently identified novel hantavirus receptor PCDH1, a cadherin superfamily member, is required for the entry of at least a subset of novel hantaviruses. Our primary objective is to generate well-characterized molecular tools to define viral and host determinants of hantavirus susceptibility and permissivity. In collaboration with Richard Yanagihara at the University of Hawaii, we will (i) generate rVSVs bearing hantavirus Gn/Gc glycoproteins representing mammalian hantavirus diversity, (ii) define the candidate hantavirus receptor requirements for cellular entry of novel hantaviruses, and (iii) finally, we will apply this knowledge to generate engineered cell lines over-expressing the relevant host factors for the isolation of authentic non-rodent-borne hantaviruses. These tools will help us achieve our long-term goals of generating a more comprehensive picture of the viral and host determinants of hantavirus susceptibility and permissivity at the molecular level.