The study of serological relationship in the nonstructural NSs protein of the genus Tospovirus

[[abstract]]The homology of nucleocapsid protein (NP) is a key criterion for demarcation of thrips-borne tospoviruses (family Bunyaviridae, genus Tospovirus) at the species level. Based on the serological and phylogenetic relationships of NPs, 19 formal and tentative tospovirus species were clustered into three major serogroups with Watermelon silver mottle virus (WSMoV), Tomato spotted wilt virus (TSWV) and Iris yellow spot virus (IYSV) as the type members. While Impatiens necrotic spot virus (INSV), Peanut yellow spot virus (PYSV) and Peanut chlorotic fan-spot virus (PCFV) were serologically distinct from any other tospoviruses and classified as serotypes. A current study reported that a monoclonal antibody (MAb) prepared to the NSs protein of WSMoV, MAb-WNSs, can broadly react with the members of both WSMoV and IYSV serogroups. Epitope mapping revealed that the MAb-WNSs-recognized region is highly conserved among the NSs proteins of those tospoviruses. Sequence analyses of the reported NSs proteins indicated that INSV shares higher degrees homology with the members of TSWV serogroup while PCFV is close to PYSV. In this investigation, the NSs proteins of INSV and PCFV were individually produced by bacterial pET expression system and used as immungens for preparation of antisera, RAs-INSs and RAs-PNSs, respectively. As expected, RAs-INSs reacted broadly with the homologous INSV antigen and the crude samples from the members of TSWV serogroup, including TSWV, Groundnut ringspot virus, Tomato chlorotic spot virus and Chrysanthemum stem necrosis virus. RAs-PNSs specifically reacted with the crude antigen of PCFV. Due to the lack of PYSV in our laboratory, serological comparison between PYSV and PCFV is unavailable at present. Nucleotide sequence corresponding to the N gene of PYSV was artificially synthesized. Furthermore, the NP of PYSV was expressed for preparation of antiserum RAs-PYSV NP. RAs-PYSV NP reacted positively with the crude sample of PCFV as well as the expressed PYSV NP. Taken together, our results of NSs serological relationships suggest that the current tospovirus species should be reclassified as four major clusters, such as WSMoV, TSWV, IYSV and PYSV serogroups. The antisera produced in this investigation are useful tools for inspection of tospoviruses