A Novel Virus of the Genus Cilevirus Causing Symptoms Similar to Citrus Leprosis

Abstract

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.

Transmisión de leprosis de los cítricos por ácaros Brevipalpus yothersi a través de hospederos no cítricos

“…Adquisición del virus por ácaros B. yothersi Para la adquisición del virus por los ácaros B. yothersi, se aislaron en un invernadero dos árboles de naranjo Valencia con lesiones del virus CiLV como fuente de inóculo, los cuales fueron previamente determinados positivos para CiLV-C y CiLV-C2 por medio de RT-PCR según Roy et al (2013). Se colectaron hojas con abundantes lesiones de la enfermedad de dichos árboles y se llevaron al laboratorio de entomología.…”

“…Para 2012, en Colombia fue detectado un nuevo tipo de virus denominado "virus de la leprosis de los cítricos citoplasmático -tipo 2" (CiLV-C2), el cual provoca síntomas muy similares a los producidos por el CiLV-C (Roy et al 2013;León et al 2014). Tras la identificación del genoma completo de este virus, se desarrolló un nuevo set de cebadores para su diagnóstico molecular por medio de RT-PCR.…”

“…León et al (2014) confirmaron que el ácaro es vector del CiLV-C2 en los departamentos del Meta y Casanare. (Roy et al 2013;2015).…”

Sequenciamento e análise da variabilidade genética de vírus transmitidos por ácaros do gênero <i>Brevipalpus </i>no Brasil

“…Hoje, o vírus está presente na maioria dos estados brasileiros e na Costa Rica (RODRIGUES et al, 2002;RAMALHO et al, 2014aRAMALHO et al, , 2015 MELZER et al, 2013a;ROY et al, 2013b). O genoma de CiLV-C2 apresenta organização semelhante à do CiLV-C, exceto pela presença de uma longa região 3` não traduzida (3`-UTR) e uma ORF extra no RNA2, que codifica uma proteína putativa de 7 kDa (MELZER et al, 2013a;ROY et al, 2013b).…”

“…No entanto, a recente caracterização molecular de novos VTB associados à leprose dos citros no México, na Colômbia e no Havaí possibilitou a disponibilização de técnicas de detecção específicas para cada VTB conhecido (MELZER et al, 2013b;CRUZ-JARAMILLO et al, 2014;ROY et al, 2014a). Inicialmente, a presença dos vírus associados à leprose dos citros (CiLV-C, CiLV-N e CiLV-C2) foi avaliada por meio de RT-PCR utilizando iniciadores específicos (LOCALI et al, 2003;ROY et al, 2013bROY et al, , 2014a. Nenhuma das 47 amostras geraram produtos de RT-PCR para CiLV-N e CiLV-C2; entretanto, todas as amostras produziram fragmentos com o tamanho esperado de 320 pb com os iniciadores específicos para detecção de CiLV-C (Figura 10).…”