A Novel Lineage of Cile-Like Viruses Discloses the Phylogenetic Continuum Across the Family Kitaviridae.

An increasing number of plant species have been recognized or considered likely reservoirs of viruses transmitted by Brevipalpus mites. A tiny fraction of these viruses, primarily those causing severe economic burden to prominent crops, have been fully characterized. In this study, based on high-throughput sequencing, transmission electron microscopy analyses of virions in plant-infected tissues, viral transmission experiments, and the morphoanatomical identification of the involved Brevipalpus mites, we describe molecular and biological features of viruses representing three new tentative species of the family Kitaviridae. The genomes of Solanum violifolium ringspot virus (SvRSV, previously partially characterized), Ligustrum chlorotic spot virus (LigCSV), and Ligustrum leprosis virus (LigLV) have five open reading frames (ORFs) > 500 nts, two distributed in RNA1 and three in RNA2. RNA1 of these three viruses display the same genomic organization found in RNA1 of typical cileviruses, while their RNA2 are shorter, possessing only orthologs of genes p61, p32, and p24. LigCSV and LigLV are more closely related to each other than to SvRSV, but the identities between their genomic RNAs were lower than 70%. In gene-by-gene comparisons, ORFs from LigCSV and LigLV had the highest sequence identity values (nt sequences: 70-76% and deduced amino acid sequences: 74-83%). The next higher identity values were with ORFs from typical cileviruses, with values below 66%. Virions of LigLV (≈ 40 nm × 55 nm) and LigCSV (≈ 54 nm × 66 nm) appear almost spherical, contrasting with the bacilliform shape of SvRSV virions (≈ 47 nm × 101 nm). Mites collected from the virus-infected plants were identified as Brevipalpus papayensis, B. tucuman, and B. obovatus. Viruliferous B. papayensis mites successfully transmitted LigCSV to Arabidopsis thaliana. SvRSV, LigCSV, and LigLV seem to represent novel sub-lineages of kitaviruses that descent on parallel evolutionary branches from a common ancestor shared with the tentative cile-like virus hibiscus yellow blotch virus and typical cileviruses. Biological and molecular data, notably, the phylogenetic reconstruction based on the RdRp proteins in which strong support for monophyly of the family Kitaviridae is observed, mark an advance in the understanding of kitavirids.

Poorly Conserved P15 Proteins of Cileviruses Retain Elements of Common Ancestry and Putative Functionality: A Theoretical Assessment on the Evolution of Cilevirus Genomes.

The genus Cilevirus groups enveloped single-stranded (+) RNA virus members of the family Kitaviridae, order Martellivirales. Proteins P15, scarcely conserved polypeptides encoded by cileviruses, have no apparent homologs in public databases. Accordingly, the open reading frames (ORFs) p15, located at the 5'-end of the viral RNA2 molecules, are considered orphan genes (ORFans). In this study, we have delved into ORFs p15 and the relatively poorly understood biochemical properties of the proteins P15 to posit their importance for viruses across the genus and theorize on their origin. We detected that the ORFs p15 are under purifying selection and that, in some viral strains, the use of synonymous codons is biased, which might be a sign of adaptation to their plant hosts. Despite the high amino acid sequence divergence, proteins P15 show the conserved motif [FY]-L-x(3)-[FL]-H-x-x-[LIV]-S-C-x-C-x(2)-C-x-G-x-C, which occurs exclusively in members of this protein family. Proteins P15 also show a common predicted 3D structure that resembles the helical scaffold of the protein ORF49 encoded by radinoviruses and the phosphoprotein C-terminal domain of mononegavirids. Based on the 3D structural similarities of P15, we suggest elements of common ancestry, conserved functionality, and relevant amino acid residues. We conclude by postulating a plausible evolutionary trajectory of ORFans p15 and the 5'-end of the RNA2 of cileviruses considering both protein fold superpositions and comparative genomic analyses with the closest kitaviruses, negeviruses, nege/kita-like viruses, and unrelated viruses that share the ecological niches of cileviruses.

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

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.

Costus stripe mosaic virus, a tentative new member of the genus Potyvirus.

Morphological, biological, serological, and molecular tests underpin the description of costus stripe mosaic virus (CoSMV) as a new member of the genus Potyvirus, family Potyviridae. Found affecting the native ornamental Costus spiralis in Brazil, the pathogen showed a severely restricted natural and experimental host range. Excluding the poly(A) tail, the CoSMV genome contains a large open reading frame (ORF) of 9,446 nucleotides that encodes a polyprotein with 3,046 amino acids, which is potentially cleaved into ten products, and a small ORF (77 amino acids) knows as PIPO. Genome analysis demonstrated the highest CoSMV nucleotide sequence identity to onion yellow dwarf virus (51.79%). No evidence of recombination was detected in the CoSMV genome, and phylogenetic analysis revealed its basal position in a group formed by members of the genus Potyvirus, along with Cyrtanthus elatus virus A (Vallota speciosa virus) and canna yellow streak virus. CoSMV was not transmitted by aphids of the species Aphis solanella, Myzus persicae or Uroleucon sonchi, which could be due to mutations in the HC-Pro motifs required for aphid transmission. A divergence in the P1 protein cleavage site was found when compared to other members of the family Potyviridae. Based on its unique biological and molecular characteristics and the current species demarcation criteria, we propose CoSMV to be a new tentative member of the genus Potyvirus.

An updated phylogenetic classification of Corynespora cassiicola isolates and a practical approach to their identification based on the nucleotide polymorphisms at the ga4 and caa5 loci.

Corynespora cassiicola (Burk. & M.A. Curtis) C.T. Wei. is an anamorphic fungus that affects more than 530 plant species, including economically important crops. Several lineages of this pathogen have been recognized, but the classification of isolates into clades is time-consuming and still sometimes leads to unclear results. In this work, eight major phylogenetic clades (PhL1-PhL8) including 245 isolates of C. cassiicola from 44 plant species were established based on a Bayesian inference analysis of four combined C. cassiicola genomic loci retrieved from GenBank, i.e., rDNA internal transcribed spacer (ITS), actin-1,ga4, and caa5. The existence of PhL1-PhL5 and PhL7 as clonal lineages was further confirmed through the analysis of full-genome single-nucleotide polymorphisms of 39 isolates. Haplotypes of the caa5 locus were PhL specific and encode isoforms of the LDB19 domain of a putative α-arrestin N-terminal-like protein. Evolution of the Caa5 arrestin is in correspondence with the PhLs. ga4 and caa5 PhL consensus sequences and a cleaved amplified polymorphic sequence (CAPS) procedure were generated based on the conserved nucleotide sequences and enzyme restriction patterns observed among isolates from the same lineage, respectively. The CAPS method was validated in silico, and its practical use allowed us to differentiate between tomato and papaya isolates, as well as to reveal the prevalence of PhL1 among isolates infecting soybean in Brazil. This novel approach could be useful in the efforts to control the diseases associated with C. cassiicola.

Dichorhaviruses in their Host Plants and Mite Vectors.

A group of related bacilliform, nuclear viruses with a bisegmented negative-sense RNA genome that are transmitted by Brevipalpus mites likely in a circulative-propagative manner were recently classified in the new genus Dichorhavirus, family Rhabdoviridae. These viruses cause localized lesions on leaves, stems, and fruits of economically significant horticultural and ornamental plant species. Among its members, orchid fleck virus, citrus leprosis virus N, and coffee ringspot virus are most prominent. This chapter summarizes the current knowledge about these viruses, available detection techniques, and their interactions with their plant hosts and mite vectors.

Making a Better Home: Modulation of Plant Defensive Response by Brevipalpus Mites.

False-spider mites of the genus Brevipalpus are highly polyphagous pests that attack hundreds of plant species of distinct families worldwide. Besides causing direct damage, these mites may also act as vectors of many plant viruses that threaten high-value ornamental plants like orchids and economically important crops such as citrus and coffee. To better understand the molecular mechanisms behind plant-mite interaction we used an RNA-Seq approach to assess the global response of Arabidopsis thaliana (Arabidopsis) plants along the course of the infestation with Brevipalpus yothersi, the main vector species within the genus. Mite infestation triggered a drastic transcriptome reprogramming soon at the beginning of the interaction and throughout the time course, deregulating 1755, 3069 and 2680 genes at 6 hours after infestation (hai), 2 days after infestation (dai), and 6 dai, respectively. Gene set enrichment analysis revealed a clear modulation of processes related to the plant immune system. Co-expressed genes correlated with specific classes of transcription factors regulating defense pathways and developmental processes. Up-regulation of defensive responses correlated with the down-regulation of growth-related processes, suggesting the triggering of the growth-defense crosstalk to optimize plant fitness. Biological processes (BPs) enriched at all time points were markedly related to defense against herbivores and other biotic stresses involving the defense hormones salicylic acid (SA) and jasmonic acid (JA). Levels of both hormones were higher in plants challenged with mites than in the non-infested ones, supporting the simultaneous induction of genes from both pathways. To further clarify the functional relevance of the plant hormonal pathways on the interaction, we evaluated the mite performance on Arabidopsis mutants impaired in SA- or JA-mediated response. Mite oviposition was lower on mutants defective in SA biosynthesis (sid2) and signaling (npr1), showing a function for SA pathway in improving the mite reproduction, an unusual mechanism compared to closely-related spider mites. Here we provide the first report on the global and dynamic plant transcriptome triggered by Brevipalpus feeding, extending our knowledge on plant-mite interaction. Furthermore, our results suggest that Brevipalpus mites manipulate the plant defensive response to render the plant more susceptible to their colonization by inducing the SA-mediated pathway.

First Complete Genome Sequence of an Isolate of Tomato Mottle Mosaic Virus Infecting Plants of Solanum lycopersicum in South America.

The complete nucleotide sequence of an isolate of tomato mottle mosaic virus (ToMMV) was determined. The virus, originally isolated from symptomatic tomato plants found in a county near the city of São Paulo, Brazil, has a genome with 99% nucleotide sequence identity with ToMMV from Mexico, China, Spain, and the United States.

Citrus leprosis virus C Infection Results in Hypersensitive-Like Response, Suppression of the JA/ET Plant Defense Pathway and Promotion of the Colonization of Its Mite Vector.

Leprosis is a serious disease of citrus caused by Citrus leprosis virus C (CiLV-C, genus Cilevirus) whose transmission is mediated by false spider mites of the genus Brevipalpus. CiLV-C infection does not systemically spread in any of its known host plants, thus remaining restricted to local lesions around the feeding sites of viruliferous mites. To get insight into this unusual pathosystem, we evaluated the expression profiles of genes involved in defense mechanisms of Arabidopsis thaliana and Citrus sinensis upon infestation with non-viruliferous and viruliferous mites by using reverse-transcription qPCR. These results were analyzed together with the production of reactive oxygen species (ROS) and the appearance of dead cells as assessed by histochemical assays. After interaction with non-viruliferous mites, plants locally accumulated ROS and triggered the salicylic acid (SA) and jasmonate/ethylene (JA/ET) pathways. ERF branch of the JA/ET pathways was highly activated. In contrast, JA pathway genes were markedly suppressed upon the CiLV-C infection mediated by viruliferous mites. Viral infection also intensified the ROS burst and cell death, and enhanced the expression of genes involved in the RNA silencing mechanism and SA pathway. After 13 days of infestation of two sets of Arabidopsis plants with non-viruliferous and viruliferous mites, the number of mites in the CiLV-C infected Arabidopsis plants was significantly higher than in those infested with the non-viruliferous ones. Oviposition of the viruliferous mites occurred preferentially in the CiLV-C infected leaves. Based on these results, we postulated the first model of plant/Brevipalpus mite/cilevirus interaction in which cells surrounding the feeding sites of viruliferous mites typify the outcome of a hypersensitive-like response, whereas viral infection induces changes in the behavior of its vector.