Serological Comparison and Molecular Characterization for Verification of Calla lily chlorotic spot virus as a New Tospovirus Species Belonging to Watermelon silver mottle virus Serogroup

Abstract

Calla lily chlorotic spot virus (CCSV) isolated from central Taiwan was recently identified as a tospovirus serologically but distantly related to Watermelon silver mottle virus (WSMoV). To clarify the serological relationship between the two viruses, rabbit polyclonal antibody (PAb) to CCSV and mouse monoclonal antibodies (MAbs) to WSMoV NP or CCSV NP were produced in this investigation, using purified nucleocapsid protein (NP) as immunogens. The PAb to CCSV NP reacted stronger with the homologous antigen than with the heterologous antigen, with much lower A405 readings in indirect enzyme-linked immunosorbent assay (ELISA) and low-intensity banding in immunoblotting. MAbs produced to CCSV NP or WSMoV NP reacted specifically with the homologous antigens but not with the heterologous antigens in both ELISA and immunoblot analyses. The CCSV S RNA was determined to be 3,172 nucleotides in length, with an inverted repeat at the 5′ and 3′ ends and two open reading frames encoding the NP and a nonstructural (NSs) protein in an ambisense arrangement. A typical 3′-terminal sequence (5′-AUUGCUCU-3′) that is shared by all members of the genus Tospovirus also is present in the CCSV S RNA. The CCSV NP and NSs protein share low amino acid identities of 20.1 to 65.1% and 19.9 to 66.1%, respectively, with those of reported tospoviruses. Phylogenetic dendrogram analysis indicates that CCSV is a distinct member in the genus Tospovirus. The results provide evidence that CCSV is a new species in the genus Tospovirus and belongs to WSMoV serogroup.