Identification and molecular characterization of nuclear Citrus leprosis virus, an unassigned Dichorhavirus genus member associated with citrus leprosis disease in Mexico

Authors: ROY, AVIJIT - University Of Florida; Stone, Andrew; Shao, Jonathan; COLINA, GABRIEL - Colegio De Postgraduados; Wei, Gang; CHOUDARY, NANDLAL - University Of Florida; ACHOR, DIANE - University Of Florida; NAKHLA, MARK - Animal And Plant Health Inspection Service (APHIS); LEVY, LAURENE - Animal And Plant Health Inspection Service (APHIS); Hartung, John; Schneider, William; BRLANSKY, RONALD - University Of Florida

Submitted to: Virus Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/18/2014
Publication Date: 4/1/2015
Citation: Roy, A., Stone, A.L., Shao, J.Y., Colina, G., Wei, G., Choudary, N., Achor, D., Nakhla, M., Levy, L., Hartung, J.S., Schneider, W.L., Brlansky, R. 2015. Identification and molecular characterization of nuclear Citrus leprosis virus, an unassigned Dichorhavirus genus member associated with citrus leprosis disease in Mexico. Virus Research. 105:564-575.

Interpretive Summary: Plants are subject to a large number of diseases, and many of these are caused by viruses. Identifying new viruses that are causing diseases can be very difficult. Most plant virus tests require significant amounts of research before the proper assay can be developed. Very few assays are capable of detecting novel or emerging plant viruses, and the few assays that are useful for discovery provide very little information about these novel viruses. This paper describes a new virus that causes a significant disease on citrus plants. The disease is called citrus leprosis, and it has been found in multiple locations in South and Central America. The most recent outbreak of citrus leprosis occurred in Mexico, and some of the virus infections found there were not the same as those found in previous outbreaks. One of these isolates was not detectable using current tests. The new, undetectable isolate was characterized by microscopy and by determining the genetic sequence. This virus is very different from the other viruses that cause leprosis, and is closely related to a virus that affects orchid. A new assay was developed that was capable of detecting the new citrus leprosis virus.

Technical Abstract: Citrus leprosis is a difficult viral disease causing significant damage to citrus fruit in South America and Central America. The disease is marked by dramatic lesions on fruit, leaves and stems resulting in unmarketable product. Citrus leprosis virus cytoplasmic type (CiLV-C) was detected in states of Chipas and Tobasco in Mexico, 2005-06. In 2011-12, CiLV affected Hamlin and Valencia oranges from Mexico were sent to the USDA-APHIS-PPQ-CPHST, Beltsville, MD, USA for testing. Leprosis samples from state of Queretaro failed to react with CiLV-C and CiLV-C type 2 (CiLV-C2) specific antisera in ELISA tests. Furthermore, in RT-PCR CiLV-C and -C2 specific primers also failed to produce amplicons. However, transmission electron microscopy of the infected leaves showed bullet shaped virions in the nuclei and cytoplasm similar to that reported for CiLV nuclear type (CiLV-N). In addition, polyclonal antibodies to Orchid Fleck Virus (OFV) reacted with extracts from infected tissues, suggesting that the leprosies symptoms could be caused by a dichorhavirus, a type of rhabdovirus. The complete genome of the new CiLV-N isolate was sequenced and compared to existing rhabdoviruses. The genome sequence was very similar to OFV in structure and sequence to OFV, but a careful phylogenetic analyses indicates that the two viruses may be different species. A reverse transcription-PCR assay was developed that could specifically identify CiLV-N, in order to better aid regulators in identifying the true cause of future citrus leprosies outbreaks.