A breakthrough in kitavirids: genetic variability, reverse-genetics, Koch’s postulates and transmission of hibiscus green spot virus 2

Abstract

Hibiscus green spot virus 2 (HGSV-2), a member of the Higrevirus genus (family Kitaviridae), is a positive-stranded RNA virus associated with leprosis-like symptoms in citrus and green spots on leaves in hibiscus. HGSV-2 has only been reported in Hawaii, and while it is speculated that mites in the genus Brevipalpus might be responsible for its transmission, proper transmission assays are yet to be conducted. This study characterizes additional citrus and hibiscus isolates of HGSV-2 collected from two Hawaiian Islands. We constructed an infectious cDNA clone from a hibiscus isolate of HGSV-2 collected on Oahu and demonstrated its ability to infect several experimental hosts, including Phaseolus vulgaris, Nicotiana tabacum, and N. benthamiana, as well as natural hosts, Citrus reticulata and Hibiscus arnottianus. Bacilliform virions with varied sizes of 33-120 nm (length) and 14-70 nm (diameter) were observed in partially purified preparations obtained from agroinoculated leaves. Virus progeny from the infectious cDNA clone was found to be infectious after mechanical transmission to N. benthamiana and cause local lesions. Lastly, an isoline colony of the mite Brevipalpus azores had vector competence to transmit a citrus isolate of HGSV-2 collected from Maui to citrus and hibiscus plants demonstrating the mite-borne nature of HGSV-2. The infectious cDNA clone developed in this study is the first reverse-genetics system for a kitavirid and will be fundamental to better characterize basic biology of HGSV-2 and its interactions with host plants and mite vectors.