Raspberries are an important commodity worldwide, with the United States (U.S.) leading fourth in global production in 2017. An important factor when shipping propagated Rubus across the globe is the security of clean plant material verified through pathogen detection methods which is vital in preventing disease spread. A raspberry virus on the quarantine list for Rubus exports from the U.S. is raspberry leaf curl virus which is a causal agent of the systemic disease known as raspberry leaf curl disease (RLCD). RLCD has been reported to be caused by two separate viruses causing varying symptoms in black, purple, and red raspberry cultivars. Although the disease has been described back in 1895 and was one of the most destructive raspberry diseases throughout the U.S. and Canada, the causal agents of the disease have not been identified. The disease is known to cause severe downward curling of the leaves, noticeably stunted shoots, leaf chlorosis, and crumbly fruit; it is the only Rubus disease that requires bioassays for detection. The objective of this study was to identify and characterize the virus complex(es) that cause disease symptoms and develop a molecular based diagnostic tool to identify them. Five viruses, black raspberry necrosis virus (BRNV), Rubus yellow net virus (RYNV) and three novel viruses, were identified in RLCD-like native Rubus and were used in single and mixed viral infections to recreate the symptoms previously described. One novel single-stranded RNA virus belonging to family Rhabdoviridae was taxonomically classified as the U.S. variant of raspberry vein chlorosis virus (RVCV) and named RVCV_US. The genome is 14,662 nucleotides (nt) in length and encodes for five structural proteins common to Rhabdoviridae and three proteins found specifically in the cytorhabdovirus genus. The genomic and phylogenetic analyses show a close evolutionary relationship of RVCV_US to Alfalfa dwarf virus (ADV) and Strawberry crinkle virus (SCV) but show a higher affinity to variants RVCV_Hutt1 and RVCV_Hutt2 isolated from the United Kingdom (U.K.). Detection primers were designed to amplify highly conserved regions of the RNA-dependent RNA polymerase (L Protein) of all three RVCV variants and were utilized in the Rubus survey to present information on the geographic distribution of the virus in the U.S. Single and mixed viral infections were performed using the large raspberry aphid, Amphorophora agathonica, and graft leaflet transmissions onto ‘Columbian’,
‘Cumberland’, and ‘Cuthbert’ raspberry cultivars. Varying symptoms such as leaf chlorosis, yellowing of minor veins, minor crinkling, and downward cupping of leaf tissue were observed in single and mixed infections throughout the experiment but RLCD symptoms were not replicated. The lack of classic symptoms previously reported on the same raspberry cultivars may be due to the exclusion of the novel Luteoviridae virus that became undetectable throughout the course of experiment despite modifying nucleic acid extraction and RT-PCR protocols.