Identification, characterisation and detection of a new tospovirus on sweet pepper

Abstract

A putative virus-induced disorder showing mottling and deformation on leaves and fruits of sweet pepper was observed in Taiwan in 2009. Crude sap of symptomatic sweet pepper leaves reacted positively in enzyme-linked immunosorbent assay (ELISA) with antisera to tospoviruses in the Watermelon silver mottle virus (WSMoV) serogroup. A virus culture, TwPep3, isolated from the symptomatic sweet pepper revealed isometric virions measuring about 80-100nm in diameter via electron microscopy. Seedlings of sweet pepper inoculated with TwPep3 showed similar symptoms as those observed in the field. A 0.9-kb viral genome of isolate TwPep3 could be amplified using degenerate primers for L RNA conserved region of tospoviruses but not using those of tobamoviruses, potyviruses and Cucumber mosaic virus. The partial L RNA sequence of isolate TwPep3 (KF383955) shared 75.1% and 82.4% nucleotide identities with those of WSMoV and Tomato necrotic ringspot virus (TNRV), respectively. To determine the taxonomic relationship of isolate TwPep3, the full length of S RNA (KF383956) was sequenced. Sequence analyses showed that the nucleocapsid (N) gene of TwPep3 shared 82-83% and 87.9-89% nucleotide and amino acid sequence identities, respectively, with those of five isolates of TNRV, the most closely related tospovirus. Moreover, the NSs proteins and S RNAs of TwPep3 and TNRV shared only 78.7% and 77.6% amino acid and nucleotide sequence identities, respectively. Based on our results, isolate TwPep3 should be considered as a new tospovirus and we propose the name Pepper chlorotic spot virus (PCSV). A specific primer pair designed in the N gene was successfully used in reverse transcription-polymerase chain reaction for PCSV diagnostic work.