Location: Molecular Plant Pathology LaboratoryTitle: A novel emerging virus with indistinguishable symptoms and genome structure similar to citrus leprosis virus C identified by small RNA deep sequencing Author
Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/14/2012
Publication Date: 12/14/2012
Publication URL: http://handle.nal.usda.gov/10113/61492
Citation: Roy, A., Choudhary, N., Govindarajulu, A., Shao, J.Y., Hartung, J.S., Brlansky, R. 2012. A novel emerging virus with indistinguishable symptoms and genome structure similar to citrus leprosis virus C identified by small RNA deep sequencing. Phytopathology. 103:488-500. Interpretive Summary: An unknown virus was discovered in Colombia that produces symptoms similar to Citrus leprosis virus (CiLV-C), and we performed research to identify that pathogen. Through the use of next generation sequencing technology the virus was determined to be a previously unknown virus in the same family as CiLV-C, and we have named it CiLV-C2. We have also developed antibody- and DNA-based assays to detect CiLV-C2. The new virus may have replaced the typical form of CiLV-C in this part of Colombia, suggesting a competitive advantage. Our work will be used by researchers and regulatory officials from the United States and Colombia to protect the citrus industry from this dangerous virus. Our results may also be used by students of the evolution of viruses.
Technical Abstract: Citrus leprosis disease (CiLD) in Colombia was previously shown to be caused by cytoplasmic Citrus leprosis virus (CiLV-C). In 2011, ELISA and RT-PCR based diagnostic methods failed to identify CiLV-C from CiLD samples, but virions similar to CiLV-C were observed in cytoplasm of the symptomatic leaves by transmission electron microscopy. A library of small RNAs was made 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 (8717 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 (5093 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. Phylogenetic analyses of the MTR, Hel, RdRp and the 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 were designed and validated to detect its presence in CiLD samples collected from the Casanare and Meta states in Colombia.