Identification and characterization of citrus yellow vein clearing virus, a putative new member of the genus Mandarivirus.

Molecular features and genomic organization were determined for Citrus yellow vein clearing virus (CYVCV), the putative viral causal agent of yellow vein clearing disease of lemon trees, reported in Pakistan, India, and more recently in Turkey and China. CYVCV isolate Y1 from Adana, Turkey, was used for deep sequencing analysis of the virus-induced small RNA fractions and for mechanical and graft inoculation of herbaceous and citrus indicator plants. A polyclonal antiserum was developed from CYVCV-Y1 purified from Phaseolus vulgaris and used in western blot assays to characterize the coat protein of CYVCV-Y1 and determine its serological relationship with related viruses. Contigs assembled from the Illumina sequenced short reads were used to construct the whole genome of Citrus yellow vein clearing virus (CYVCV), consisting in a positive-sense RNA of 7,529 nucleotides and containing six predicted open reading frames. The CYVCV genome organization and size resembled that of flexiviruses, and search for sequence homologies revealed that Indian citrus ringspot virus (ICRSV) (Mandarivirus, Alphaflexiviridae) is the most closely related virus. However, CYVCV had an overall nucleotide sequence identity of ≈74% with ICRSV. Although the two viruses were similar with regard to genome organization, viral particles, and herbaceous host range, CYVCV caused different symptoms in citrus and was serologically distinct from ICRSV. Primer pairs were designed and used to detect the virus by conventional and quantitative reverse transcription-polymerase chain reaction on yellow vein clearing symptomatic field trees as well as graft- and mechanically inoculated host plants. Collectively, these data suggest that CYVCV is the causal agent of yellow vein clearing disease and represents a new species in the genus Mandarivirus.

Production of a monoclonal antibody specific to the El Amar strain of plum pox virus.

Plum pox virus (PPV) isolates are grouped into three clusters differentiated by biological, serological, molecular and epidemiological characteristics: Marcus (M), Dideron (D) and Cherry (C). The El Amar (EA) isolate that does not fit any of the above groups is also known. Monoclonal antibodies (MAbs) that specifically recognize M, D, and C strains of PPV are already available. To complete the set of PPV strain-specific serological reagents, MAbs against the EA isolate were raised by immunizing BALB/c mice and fusing their spleen cells with NS0/1 myeloma cells. After a preliminary characterization by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), 1 of 13 selected MAbs proved to be EA strain-specific. This MAb (EA24) reacted equally well with a homologous antigen and several PPV isolates from Egyptian apricot trees, supporting the hypothesis of an additional specific PPV group. MAb EA24 did not react either with about a hundred PPV isolates belonging to the D and M groups or with PPV-SwC and PPV-SoC isolates belonging to the C group. The strain specificity of MAb EA24 was confirmed by Western blot analysis and immunoelectron microscopy. We conclude that there is now available a set of MAbs which are highly specific to the four currently known groups of PPV strains.