title: Identification and Molecular Characterization of Nuclear Citrus leprosis virus, a Member of the Proposed Dichorhavirus Genus Infecting Multiple Citrus Species in Mexico.
abstract: Citrus leprosis is one of the most destructive diseases of Citrus spp. and is associated with two unrelated virus groups that produce particles primarily in either the cytoplasm or nucleus of infected plant cells. Symptoms of leprosis, including chlorotic spots surrounded by yellow haloes on leaves and necrotic spots on twigs and fruit, were observed on leprosis-affected mandarin and navel sweet orange trees in the state of Querétaro, Mexico. Serological and molecular assays showed that the cytoplasmic types of Citrus leprosis virus (CiLV-C) often associated with leprosis symptomatic tissues were absent. However, using transmission electron microscopy, bullet-shaped rhabdovirus-like virions were observed in the nuclei and cytoplasm of the citrus leprosis-infected leaf tissues. An analysis of small RNA populations from symptomatic tissue was carried out to determine the genome sequence of the rhabdovirus-like particles observed in the citrus leprosis samples. The complete genome sequence showed that the nuclear type of CiLV (CiLV-N) present in the samples consisted of two negative-sense RNAs: 6,268-nucleotide (nt)-long RNA1 and 5,847-nt-long RNA2, excluding the poly(A) tails. CiLV-N had a genome organization identical to that of Orchid fleck virus (OFV), with the exception of shorter 5' untranslated regions in RNA1 (53 versus 205 nt) and RNA2 (34 versus 182 nt). Phylogenetic trees constructed with the amino acid sequences of the nucleocapsid (N) and glycoproteins (G) and the RNA polymerase (L protein) showed that CiLV-N clusters with OFV. Furthermore, phylogenetic analyses of N protein established CiLV-N as a member of the proposed genus Dichorhavirus. Reverse-transcription polymerase chain reaction primers for the detection of CiLV-N were designed based on the sequence of the N gene and the assay was optimized and tested to detect the presence of CiLV-N in both diseased and symptom-free plants.
authors.affiliation: First, sixth, seventh, and twelfth authors: University of Florida, IFAS, Plant Pathology Department, Citrus Research and Education Center, 700 Experiment Station Road, Lake Alfred, FL; second and eleventh authors: United States Department of Agriculture-Agricultural Research Service (USDA-ARS), Foreign Disease-Weed Science Research Unit (FDWSRU), Fort Detrick, MD; third and tenth authors: USDA-ARS, Molecular Plant Pathology Laboratory (MPPL), Beltsville, MD; fourth author: Colegio de Postgraduados, Campus Montecillo, Texcoco, Edo. De Mex., CP 56230, México; fifth and ninth authors: USDA-Animal and Plant Health Inspection Service (APHIS)-Plant Protection and Quarantine (PPQ)-Center for Plant Health Science and Technology (CSIRO), Beltsville, MD; and eighth author: USDA-APHIS-PPQ-CPHST, Riverdale, MD.
authors.forename: Avijit
authors.initials: A
authors.lastname: Roy
authors.forename: Andrew L
authors.initials: AL
authors.lastname: Stone
authors.forename: Jonathan
authors.initials: J
authors.lastname: Shao
authors.forename: Gabriel
authors.initials: G
authors.lastname: Otero-Colina
authors.forename: Gang
authors.initials: G
authors.lastname: Wei
authors.forename: Nandlal
authors.initials: N
authors.lastname: Choudhary
authors.forename: Diann
authors.initials: D
authors.lastname: Achor
authors.forename: Laurene
authors.initials: L
authors.lastname: Levy
authors.forename: Mark K
authors.initials: MK
authors.lastname: Nakhla
authors.forename: John S
authors.initials: JS
authors.lastname: Hartung
authors.forename: William L
authors.initials: WL
authors.lastname: Schneider
authors.forename: Ronald H
authors.initials: RH
authors.lastname: Brlansky
mesh_terms: D000595:Amino Acid Sequence; D002957:Citrus / Q000821:virology*; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D005638:Fruit / Q000821:virology; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing; D008800:Mexico; D008969:Molecular Sequence Data; D019251:Nucleocapsid / Q000235:genetics; D010802:Phylogeny; D010935:Plant Diseases / Q000821:virology*; D018515:Plant Leaves / Q000821:virology; D010942:Plant Viruses / Q000145:classification* / Q000235:genetics / Q000648:ultrastructure; D012328:RNA Viruses / Q000145:classification* / Q000235:genetics / Q000648:ultrastructure; D012367:RNA, Viral / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D014771:Virion
chemical_list: D018076:DNA, Complementary; D012367:RNA, Viral