Thottapalayam virus (TPMV), a member of the genus Hantavirus in the family Bunyaviridae, was isolated from an insectivore, Suncus murinus (musk shrew), captured in southern India in 1964. While the isolation of TPMV predates the discovery of the prototype Hantaan virus, little is known about its genetics and biology. To date, preliminary evidence suggests that TPMV differs significantly, both antigenically and genetically, from all known rodent-borne hantaviruses. However, since detailed epizootiological studies have not been conducted, it is unclear if TPMV is naturally harbored by an insectivore host or if TPMV represents a "spillover" from its natural rodent reservoir host. Moreover, to what extent TPMV causes infection and/or disease in humans is not known. To address these issues, we first studied the antigenic profile of TPMV using monoclonal antibodies against Hantaan and Seoul viruses and polyclonal immune sera against Puumala virus and TPMV. Armed with this newfound information, we developed an enzyme-linked immunosorbent assay system for the diagnosis of TPMV infections in shrews and humans, using a recombinant TPMV N antigen manipulated to have an E5/G6 epitope to be captured by monoclonal antibody clone E5/G6. Using this assay, we found anti-TPMV antibodies in sera from a patient with high fever of unknown etiology in Thailand and from two shrews captured in Indonesia. Seropositivity was verified by the indirect immunofluorescence antibody test, Western blotting analysis, and focus reduction neutralization test. Collectively, our data indicate that TPMV is harbored by Suncus murinus as its host in nature and is capable of infecting humans.