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Virome analyses of Hevea brasiliensis using small RNA deep sequencing and PCR techniques reveal the presence of a potential new virus
Authors:Paula L. C. Fonseca  Fernanda Badotti  Tatiana F. P. de Oliveira  Antônio Fonseca  Aline B. M. Vaz  Luiz M. R. Tomé  Jônatas S. Abrahão  João T. Marques  Giliane S. Trindade  Priscila Chaverri  Eric R. G. R. Aguiar  Aristóteles Góes-Neto
Affiliation:1.Department of Microbiology, Instituto de Ciências Biológicas,Universidade Federal de Minas Gerais (UFMG),Belo Horizonte,Brazil;2.Department of Chemistry, Centro Federal de Educa??o Tecnológica de Minas Gerais (CEFET-MG),Belo Horizonte,Brazil;3.LANAGRO/MG –Laboratório Nacional da Agricultura, Ministério da Agricultura (MAPA),Pedro Leopoldo,Brazil;4.Faculdade de Minas (FAMINAS),Belo Horizonte,Brazil;5.Department of Biochemistry and Immunology, Instituto de Ciências Biológicas,Universidade Federal de Minas Gerais (UFMG),Belo Horizonte,Brazil;6.Department of Plant Science and Landscape Architecture,University of Maryland,College Park,USA;7.Escuela de Biología, Universidad de Costa Rica, San Pedro,San José,Costa Rica;8.Instituto de Ciências da Saúde, Universidade Federal da Bahia (UFBA),Salvador,Brazil
Abstract:

Background

Hevea brasiliensis is an important commercial crop due to the high quality of the latex it produces; however, little is known about viral infections in this plant. The only virus described to infect H. brasiliensis until now is a Carlavirus, which was described more than 30?years ago. Virus-derived small interfering RNA (vsiRNAs) are the product of the plant’s antiviral defense triggered by dsRNA viral intermediates generated, during the replication cycle. These vsiRNAs are complementar to viral genomes and have been widely used to identify and characterize viruses in plants.

Methods

In the present study, we investigated the virome of leaf and sapwood samples from native H. brasiliensis trees collected in two geographic areas in the Brazilian Amazon. Small RNA (sRNA) deep sequencing and bioinformatic tools were used to assembly, identify and characterize viral contigs. Subsequently, PCR amplification techniques were performed to experimentally verify the presence of the viral sequences. Finally, the phylogenetic relationship of the putative new virus with related viral genomes was analyzed.

Results

Our strategy allowed the identification of 32 contigs with high similarity to viral reference genomes, from which 23 exhibited homology to viruses of the Tymoviridae family. The reads showed a predominant size distribution at 21?nt derived from both strands, which was consistent with the vsiRNAs profile. The presence and genome position of the viral contigs were experimentally confirmed using droplet digital PCR amplifications. A 1913 aa long fragment was obtained and used to infer the phylogenetic relationship of the putative new virus, which indicated that it is taxonomically related to the Grapevine fleck virus, genus Maculavirus. The putative new virus was named Hevea brasiliensis virus (HBrV) in reference to its host.

Conclusion

The methodological strategy applied here proved to be efficient in detecting and confirming the presence of new viral sequences on a ‘very difficult to manage’ sample. This is the second time that viral sequences, that could be ascribed as a putative novel virus, associated to the rubber tree has been identified.
Keywords:
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