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Molecular Epidemiology of Citrus Leprosis Virus C: A New Viral Lineage and Phylodynamic of the Main Viral Subpopulations in the Americas - PubMed

  • ️Fri Jan 01 2021

Molecular Epidemiology of Citrus Leprosis Virus C: A New Viral Lineage and Phylodynamic of the Main Viral Subpopulations in the Americas

Camila Chabi-Jesus et al. Front Microbiol. 2021.

Abstract

Despite the importance of viral strains/variants as agents of emerging diseases, genetic and evolutionary processes affecting their ecology are not fully understood. To get insight into this topic, we assessed the population and spatial dynamic parameters of citrus leprosis virus C (CiLV-C, genus Cilevirus, family Kitaviridae). CiLV-C is the etiological agent of citrus leprosis disease, a non-systemic infection considered the main viral disorder affecting citrus orchards in Brazil. Overall, we obtained 18 complete or near-complete viral genomes, 123 complete nucleotide sequences of the open reading frame (ORF) encoding the putative coat protein, and 204 partial nucleotide sequences of the ORF encoding the movement protein, from 430 infected Citrus spp. samples collected between 1932 and 2020. A thorough examination of the collected dataset suggested that the CiLV-C population consists of the major lineages CRD and SJP, unevenly distributed, plus a third one called ASU identified in this work, which is represented by a single isolate found in an herbarium sample collected in Asuncion, Paraguay, in 1937. Viruses from the three lineages share about 85% nucleotide sequence identity and show signs of inter-clade recombination events. Members of the lineage CRD were identified both in commercial and non-commercial citrus orchards. However, those of the lineages SJP were exclusively detected in samples collected in the citrus belt of São Paulo and Minas Gerais, the leading Brazilian citrus production region, after 2015. The most recent common ancestor of viruses of the three lineages dates back to, at least, ∼1500 years ago. Since citrus plants were introduced in the Americas by the Portuguese around the 1520s, the Bayesian phylodynamic analysis suggested that the ancestors of the main CiLV-C lineages likely originated in contact with native vegetation of South America. The intensive expansion of CRD and SJP lineages in Brazil started probably linked to the beginning of the local citrus industry. The high prevalence of CiLV-C in the citrus belt of Brazil likely ensues from the intensive connectivity between orchards, which represents a potential risk toward pathogen saturation across the region.

Keywords: Brevipalpus mites; Cilevirus; Kitaviridae; citrus leprosis disease; virus evolution.

Copyright © 2021 Chabi-Jesus, Ramos-González, Postclam-Barro, Fontenele, Harakava, Bassanezi, Moreira, Kitajima, Varsani and Freitas-Astúa.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1

Symptoms of citrus leprosis (CL) disease and place of collection of the infected Citrus spp. samples across Latin America. (A,B,D,F) Samples conserved at the Herbarium of Instituto Biológico, São Paulo, Brazil. (C,E,G) Fresh citrus samples. (A) CiLV-C isolate Jbt02, Jaboticabal, São Paulo, Brazil, 1975. (B) CiLV-C isolate Urg01, Uruguaiana, Rio Grande do Sul, Brazil, 1937. (C) CiLV-C isolate Bar25, São Paulo, Brazil, 2018. (D) CiLV-C isolate Asu02 from Asunción, Paraguay, 1937. (E) CiLV-C isolate PY03, Paraguay, 2010 (fruit) and 2015 (leaf). (F) CiLV-C isolate Ar06, Misiones, Argentina, 1937. (G) CiLV-C isolate AR04, Corrientes, Argentina, 2017.

FIGURE 2
FIGURE 2

Similarity plots of the nucleotide sequences of the RNA1 and RNA2 of the CiLV-C isolates. The full-length genome sequences of the 18 studied CiLV-C isolates were compared to those of the type-member viruses of the clades CRD (A) and SJP (B). Curves depict the comparison between the analyzed and a reference genome. Each plotted point is the percent identity (vertical axis) within a sliding window 200 bp wide centered on the position plotted, with a step size between points of 20 bp. The horizontal axis indicates the nucleotide positions across the RNA1 and RNA2 molecules of the reference genomes. The horizontal bars above the curves are a cartoon of the ORFs of the CiLV-C genome. Plots were generated using SimPlot (Lole et al., 1999).

FIGURE 3
FIGURE 3

Evidence of recombination in the RNA2 of CiLV-C. The diagram depicts three recombination events detected using the alignment of the completed RNA2 molecules of 23 isolates. Multicolored horizontal bars represent the genome of the recombinant molecules. Major and minor parentals are indicated by black and gray bars and numbers (#) as described in the legend. Dashed vertical lines indicate the recombination breakpoints using a cartoon representing the RNA2 molecule of CiLV-C as reference. Event 1 shows the recombination found in all isolates of the clade SJP, event 2 was detected in the isolate PY_Asu02 (clade ASU), and event 3 in BR_SP_SJP01 (clade SJP). Recombination events were detected by more than four programs implemented in the software RDP. Details of these and other recombination events are shown in Supplementary Table 4.

FIGURE 4
FIGURE 4

Phylogenetic relationships and genetic distances among CiLV-C strains. Phylogenetic trees were generated by Bayesian inference using MrBayes and based on p29 (190 isolates, 795 nts) and p32 (270 isolates, 288 nts, partial) (A), and the complete sequences of the RNA1 molecules and partial sequences of the RNA2 molecules (p61, p32, and p24) of 23 isolates of CiLV-C (B). Inter- and intra-clade nucleotide distances were calculated considering independent ORFs and complete genomic molecules (C). The maximum clade credibility trees were generated with 6,000,000 generations and using the appropriate sequences of CiLV-C2_Colombia (NC038848 and NC038849) as an outgroup. Tree branches were identified according to CiLV-C lineages.

FIGURE 5
FIGURE 5

Distribution of CiLV-C strains in Latin America (A) and across municipalities in the Brazilian states of São Paulo and Minas Gerais (B) in the period from 1932 to 2020. Viral detection was based on RT-PCR assays for specific identification of p29 and p24 from isolates of the CRD and SJP clade. CiLV-C_ASU was identified by high-throughput sequencing. The color-coded legend indicates the presence of a viral isolate, concomitantly or at a different time in the same plant, in single or mixed infections. The map also depicts the distribution of CiLV-C isolates whose sequences were already available in the GenBank database.

FIGURE 6
FIGURE 6

Bayesian maximum-clade-credibility time-scaled phylogenetic tree using the concatenated sequences of the p29 (795 nts) and p32 (288 nts, partial) ORFs from 132 CiLV-C isolates collected in the period 1932–2020 in South America. Horizontal gray bars on nodes (A–D) indicate the uncertainty for the date of each node (95% highest posterior density—HPD—intervals). Figures near the main nodes represent the posterior probability values. The phylogenetic tree was edited using IcyTree.

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