Kitaviruses: A Window to Atypical Plant Viruses Causing Nonsystemic Diseases.

Kitaviridae is a family of plant-infecting viruses that have multiple positive-sense, single-stranded RNA genomic segments. Kitaviruses are assigned into the genera Cilevirus, Higrevirus, and Blunervirus, mainly on the basis of the diversity of their genomic organization. Cell-to-cell movement of most kitaviruses is provided by the 30K family of proteins or the binary movement block, considered an alternative movement module among plant viruses. Kitaviruses stand out for producing conspicuously unusual locally restricted infections and showing deficient or nonsystemic movement likely resulting from incompatible or suboptimal interactions with their hosts. Transmission of kitaviruses is mediated by mites of many species of the genus Brevipalpus and at least one species of eriophyids. Kitavirus genomes encode numerous orphan open reading frames but RNA-dependent RNA polymerase and the transmembrane helix-containing protein, generically called SP24, typify a close phylogenetic link with arthropod viruses. Kitaviruses infect a large range of host plants and cause diseases of economic concern in crops such as citrus, tomato, passion fruit, tea, and blueberry.

History and Diversity of Citrus leprosis virus Recorded in Herbarium Specimens.

Leprosis refers to two diseases of citrus that present similar necrotic local lesions, often surrounded by chlorotic haloes on citrus. Two distinct viruses are associated with this disease, one that produces particles primarily in the nucleus of infected plant cells (Citrus leprosis virus nuclear type [CiLV-N]; Dichorhavirus) and another type that produces particles in the cytoplasm of infected plant cells (Citrus leprosis virus cytoplasmic type [CiLV-C]; Cilevirus). Both forms are transmitted by Brevipalpid mites and have bipartite, single-stranded, RNA genomes. CiLV-C and CiLV-N are present in South and Central America and as far north as parts of Mexico. Although leprosis disease was originally described from Florida, it disappeared from there in the 1960s. The United States Department of Agriculture-Agricultural Research Service maintains preserved citrus specimens identified at inspection stations 50 or more years ago with symptoms of citrus leprosis. We isolated RNA from these samples and performed degradome sequencing. We obtained nearly full-length genome sequences of both a typical CiLV-C isolate intercepted from Argentina in 1967 and a distinct CiLV-N isolate obtained in Florida in 1948. The latter is a novel form of CiLV-N, not known to exist anywhere in the world today. We have also documented the previously unreported presence of CiLV-N in Mexico in the mid-20th century.

A novel virus of the genus Cilevirus causing symptoms similar to citrus leprosis.

Citrus leprosis in Colombia was previously shown to be caused by cytoplasmic Citrus leprosis virus (CiLV-C). In 2011, enzyme-linked immunosorbent assay and reverse-transcription polymerase chain reaction (RT-PCR)-based diagnostic methods failed to identify CiLV-C from citrus samples with symptoms similar to citrus leprosis; however, virions similar to CiLV-C were observed in the cytoplasm of the symptomatic leaves by transmission electron microscopy. Furthermore, the causal organism was transmitted by the false spider mite, Brevipalpus phoenicis, to healthy citrus seedlings. A library of small RNAs was constructed from symptomatic leaves and used as the template for Illumina high-throughput parallel sequencing. The complete genome sequence and structure of a new bipartite RNA virus was determined. RNA1 (8,717 nucleotides [nt]) contained two open reading frames (ORFs). ORF1 encoded the replication module, consisting of five domains: namely, methyltransferase (MTR), cysteine protease-like, FtsJ-MTR, helicase (Hel), and RNA-dependent RNA polymerase (RdRp); whereas ORF2 encoded the putative coat protein. RNA2 (4,989 nt) contained five ORFs that encode the movement protein (MP) and four hypothetical proteins (p7, p15, p24, and p61). The structure of this virus genome resembled that of CiLV-C except that it contained a long 3' untranslated terminal region and an extra ORF (p7) in RNA2. Both the RNA1 and RNA2 of the new virus had only 58 and 50% nucleotide identities, respectively, with known CiLV-C sequences and, thus, it appears to be a novel virus infecting citrus. Phylogenetic analyses of the MTR, Hel, RdRp, and MP domains also indicated that the new virus was closely related to CiLV-C. We suggest that the virus be called Citrus leprosis virus cytoplasmic type 2 (CiLV-C2) and it should be unambiguously classified as a definitive member of the genus Cilevirus. A pair of CiLV-C2 genome-specific RT-PCR primers was designed and validated to detect its presence in citrus leprosis samples collected from the Casanare and Meta states in Colombia.

Characterization of a virus infecting Citrus volkameriana with citrus leprosis-like symptoms.

A Citrus volkameriana tree displaying symptoms similar to citrus leprosis on its leaves and bark was found in Hawaii. Citrus leprosis virus C (CiLV-C)-specific detection assays, however, were negative for all tissues tested. Short, bacilliform virus-like particles were observed by transmission electron microscopy in the cytoplasm of symptomatic leaves but not in healthy controls. Double-stranded (ds) RNAs ≈8 and 3 kbp in size were present in symptomatic leaf tissue but not in healthy controls. Excluding poly(A) tails, the largest molecule, RNA1, was 8,354 bp in length. The ≈3 kbp dsRNA band was found to be composed of two distinct molecules, RNA2 and RNA3, which were 3,169 and 3,113 bp, respectively. Phylogenetic analyses indicated that the RNA-dependent RNA polymerase (RdRp) domain located in RNA1 was most closely related to the RdRp domain of CiLV-C. A reverse-transcription polymerase chain reaction assay developed for the detection of this virus was used to screen nearby citrus trees as well as Hibiscus arnottianus plants with symptoms of hibiscus green spot, a disease associated with infection by Hibiscus green spot virus (HGSV). All nearby citrus trees tested negative with the assay; however, symptomatic H. arnottianus plants were positive. All three RNAs were present in symptomatic H. arnottianus and were >98% identical to the RNAs isolated from C. volkameriana. We contend that the virus described in this study is HGSV, and propose that it be the type member of a new virus genus, Higrevirus.

Vírus da leprose dos citros: sequenciamento completo do genoma, diversidade genética e avaliação de genes potencialmente relacionados com patogenicidade / Citrus leprosis virus: genome sequencing, genetic diversity and evaluation of pathogenicity related genes

A partir das primeiras sequências genômicas do Cil V-C (Citrus leprosis virus ­tipo citoplasmático) através de bibliotecas de cDNA provenientes do produto da replicação viral (dsRNA) (locali, 2002), foi possível concluir o seqüenciamento completo do seu genoma. As informações obtidas possibilitaram caracterizá-lo como um vírus de genoma constituído por ssRNA (mais), bipartido (menos 14 Kb), contendo no RNA 1 duas ORFs, uma delas codificadora de uma poliproteína com três domínios envolvidos com replicase viral e um relacionado com protease. No RNA 2, foram detectadas quatro ORFs, uma delas codificadora da proteína de movimento. Foram identificadas caudas poli A nas extremidades 3 dos dois RNAs, bem como a presença de uma sequência conservada de nucleotídeos (GAUAAAUCU) nas extremidades 5 dos dois RNAs, sugerindo a presença de uma estrutura Cap. As informações até então conhecidas sobre o vírus associado à leprose dos citros e aos ácaros do gênero Brevipalpus os relacionavam à família Rhabdoviridae. Entretanto, através de análises estruturais e organizacionais do genoma do CilV-C ficou evidente que o vírus apresenta alguns (poucos) domínios conservados com membros de vários gêneros e famílias de fitovírus, mas não com rhabdovírus. Através de análises filogenéticas e associando estas informações às características morfológicas do virion, além dos efeitos citopáticos e sintomas induzidos por esses vírus em seus hospedeiros, sugerimos que o CilV-C seja considerado o membro-tipo de um novo gênero de vírus, denominado Cilevirus. Foi possível também determinar com segurança que ele não pertence a família Rhabdoviridae, como proposto anteriormente. Essas informações permitiram uma análise comparativa entre regiões genômicas do CilV-C com as de outros vírus transmitidos por Brevipa/pus (VTBs), entre eles, Orchid fleck virus - OFV, Coffee ringspot virus - CoRSV, e Ligustrum ringspot virus - LigRSV.