Chikungunya virus produced by a persistently infected mosquito cell line comprises a shorter genome and is non-infectious to mammalian cells.

Although RNA viruses have high mutation rates, host cells and organisms work as selective environments, maintaining the viability of virus populations by eliminating deleterious genotypes. In serial passages of RNA viruses in a single cell line, most of these selective bottlenecks are absent, with no virus circulation and replication in different tissues or host alternation. In this work, Aedes aegypti Aag-2 cells were accidentally infected with Chikungunya virus (CHIKV) and Mayaro virus (MAYV). After numerous passages to achieve infection persistency, the infectivity of these viruses was evaluated in Ae. albopictus C6/36 cells, African green monkey Vero cells and primary-cultured human fibroblasts. While these CHIKV and MAYV isolates were still infectious to mosquito cells, they lost their ability to infect mammalian cells. After genome sequencing, it was observed that CHIKV accumulated many nonsynonymous mutations and a significant deletion in the coding sequence of the hypervariable domain in the nsP3 gene. Since MAYV showed very low titres, it was not sequenced successfully. Persistently infected Aag-2 cells also accumulated high loads of short and recombinant CHIKV RNAs, which seemed to have been originated from virus-derived DNAs. In conclusion, the genome of this CHIKV isolate could guide mutagenesis strategies for the production of attenuated or non-infectious (to mammals) CHIKV vaccine candidates. Our results also reinforce that a paradox is expected during passages of cells persistently infected by RNA viruses: more loosening for the development of more diverse virus genotypes and more pressure for virus specialization to this constant cellular environment.

Low virus diversity and spread in wild Capsicum spp. accessions from Ecuador under natural inoculum pressure.

Challenging wild plant accessions with pathogens is an initial approach for finding resistance genes for breeding programs. Viruses can be transmitted artificially by mechanical or arthropod-borne inoculation, but these experimental assays do not always reproduce natural conditions in the field. In this study, 56 wild Capsicum spp. accessions from Ecuador that were under natural inoculum pressure for six months were screened for virus infections by RNA sequencing. These plants exhibited low virus diversity in comparison to a commercial pepper cultivar that was used as a susceptible host. Subjecting numerous plants to natural infection prior to artificial assays may indicate promising accessions to track within virus/vector resistance breeding programs.

Tobamoviruses of two new species trigger resistance in pepper plants harbouring functional L alleles.

Tobamoviruses are often referred to as the most notorious viral pathogens of pepper crops. These viruses are not transmitted by invertebrate vectors, but rather by physical contact and seeds. In this study, pepper plants displaying mild mottle and mosaic symptoms were sampled in four different regions of Peru. Upon double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) tests, seven samples cross-reacted weakly with antibodies against pepper mild mottle virus (PMMoV), suggesting the presence of tobamoviruses. When employing RT-PCR, conserved primers amplified cDNA fragments of viruses from two putative new tobamovirus species in the samples. The complete genome of two representative isolates were, therefore, sequenced and analysed in silico. These viruses, which were tentatively named yellow pepper mild mottle virus (YPMMoV) and chilli pepper mild mottle virus (CPMMoV), shared highest nucleotide genome sequence identities of 83 and 85 % with bell pepper mottle virus (BpeMV), respectively. Mechanical inoculation of indicator plants with YPMMoV and CPMMoV isolates did not show any obvious differences in host ranges. These viruses were also inoculated mechanically on pepper plants harbouring different resistance L alleles to determine their pathotypes. Pepper plants carrying unfunctional L alleles (L0) to tobamoviruses were infected by all isolates and presented differential symptomatology for YPMMoV and CPMMoV. On the other hand, pepper plants carrying L1, L2, L3 and L4 alleles were resistant to all isolates, indicating that these viruses belong to pathotype P0.

Molecular Characterization of Hovenia Dulcis-Associated Virus 1 (HDaV1) and 2 (HDaV2): New Tentative Species within the Order Picornavirales.

In a systematic field survey for plant-infecting viruses, leaf tissues were collected from trees showing virus-like symptoms in Brazil. After viral enrichment, total RNA was extracted and sequenced using the MiSeq platform (Illumina). Two nearly full-length picorna-like genomes of 9534 and 8158 nucleotides were found associated with Hovenia dulcis (Rhamnaceae family). Based upon their genomic information, specific primers were synthetized and used in RT-PCR assays to identify plants hosting the viral sequences. The larger contig was tentatively named as Hovenia dulcis-associated virus 1 (HDaV1), and it exhibited low nucleotide and amino acid identities with Picornavirales species. The smaller contig was related to insect-associated members of the Dicistroviridae family but exhibited a distinct genome organization with three non-overlapping open reading frames (ORFs), and it was tentatively named as Hovenia dulcis-associated virus 2 (HDaV2). Phylogenetic analysis using the amino acid sequence of RNA-dependent RNA polymerase (RdRp) revealed that HDaV1 and HDaV2 clustered in distinct groups, and both viruses were tentatively assigned as new members of the order Picornavirales. HDaV2 was assigned as a novel species in the Dicistroviridae family. The 5' ends of both viruses are incomplete. In addition, a nucleotide composition analysis (NCA) revealed that HDaV1 and HDaV2 have similarities with invertebrate-infecting viruses, suggesting that the primary host(s) of these novel virus species remains to be discovered.

A new virus found in garlic virus complex is a member of possible novel genus of the family Betaflexiviridae (order Tymovirales).

Plant vegetative propagation strategies for agricultural crops cause the accumulation of viruses, resulting in the formation of virus complexes or communities. The cultivation of garlic is based on vegetative propagation and more than 13 virus species from the genera Potyvirus, Allexivirus and Carlavirus have been reported. Aiming for an unbiased identification of viruses from a garlic germplasm collection in Brazil, total RNA from eight garlic cultivars was sequenced by high-throughput sequencing (HTS) technology. Although most viruses found in this study were previously reported, one of them did not belong to any known genera. This putative new virus was found in seven out of eight garlic cultivars and phylogenetic data positioned it as representative of an independent evolutionary lineage within family Betaflexiviridae. This virus has been tentatively named garlic yellow mosaic-associated virus (GYMaV), sharing highest nucleotide identities with African oil palm ringspot virus (genus Robigovirus) and potato virus T (genus Tepovirus) for the replicase gene, and with viruses classified within genus Foveavirus for the coat protein gene. Due to its high frequency in garlic cultivars, GYMaV should be considered in upcoming surveys of pathogens in this crop and in the development of virus-free garlic plants.

The Sw-5 Gene Cluster: Tomato Breeding and Research Toward Orthotospovirus Disease Control.

The Sw-5 gene cluster encodes protein receptors that are potentially able to recognize microbial products and activate signaling pathways that lead to plant cell immunity. Although there are several Sw-5 homologs in the tomato genome, only one of them, named Sw-5b, has been extensively studied due to its functionality against a wide range of (thrips-transmitted) orthotospoviruses. The Sw-5b gene is a dominant resistance gene originally from a wild Peruvian tomato that has been used in tomato breeding programs aiming to develop cultivars with resistance to these viruses. Here, we provide an overview starting from the first reports of Sw-5 resistance, positional cloning and the sequencing of the Sw-5 gene cluster from resistant tomatoes and the validation of Sw-5b as the functional protein that triggers resistance against orthotospoviruses. Moreover, molecular details of this plant-virus interaction are also described, especially concerning the roles of Sw-5b domains in the sensing of orthotospoviruses and in the signaling cascade leading to resistance and hypersensitive response.

High-throughput sequencing reveals a novel closterovirus in arracacha (Arracacia xanthorrhiza).

High-throughput sequencing analysis detected a clostero-like virus from arracacha plants (Arracacia xanthorrhiza) in Brazil. The complete genome sequence, confirmed by RACE and Sanger sequencing, consists of 15,763 nucleotides with nine predicted open reading frames (ORFs) in a typical closterovirus genome organisation. The putative RNA-dependent RNA polymerase (RdRp), heat shock protein 70 homologue (Hsp70h), and coat protein showed 55-65, 38-44, and 20-36% amino acid sequence identity, respectively, to the homologous proteins of known closteroviruses. Phylogenetic analysis of Hsp70h showed that this putative novel arracacha plant virus was related to members of the genus Closterovirus in the family Closteroviridae. These results suggest that this virus, tentatively named "arracacha virus 1" (AV-1), is a novel member of the genus Closterovirus. This is the first closterovirus identified in arracacha plants.

Plant responses to tomato chlorotic mottle virus: Proteomic view of the resistance mechanisms to a bipartite begomovirus in tomato.

Tomato chlorotic mottle virus (ToCMoV) is a widespread bipartite Begomovirus species found in tomato fields in Brazil. In this study, plant responses and putative mechanisms associated with the 'Tyking'-derived recessive resistance to ToCMoV were investigated. Changes in the protein profile in the inoculated plants of two near isogenic tomato lines resistant ('LAM 157') and susceptible ('Santa Clara') to ToCMoV were analyzed. Seedlings were biolistically inoculated with an infectious ToCMoV clone. Leaves from infected plants (confirmed by PCR) were sampled at 15days after inoculation. Proteins were extracted using phenol and analyzed by shotgun MS (2D-nanoUPLC/HDMSE). Out of the 534 identified proteins, 82 presented statistically significant differences in abundance, including 35 unique proteins displayed in the resistant tomato inoculated with ToCMoV. Proteins associated to chromatin structure, cytoskeleton structure, cuticle biosynthesis, and ubiquitin pathway were identified and their putative roles during virus infection process were discussed. The protein profile analysis allowed for the development of a hypothetical model showing how the resistant host cell responds to ToCMoV infection. The data obtained provide a better understanding of resistant mechanisms used by the host plant to contain viral infection and could be the basis for further investigation in other plant-begomovirus pathosystems. BIOLOGICAL SIGNIFICANCE: In this study we propose a model of resistance to begomovirus in tomato and highlight host proteins, which could be targets for future investigations in plant-begomovirus pathosystems.

The functional analysis of distinct tospovirus movement proteins (NSM) reveals different capabilities in tubule formation, cell-to-cell and systemic virus movement among the tospovirus species.

The lack of infectious tospovirus clones to address reverse genetic experiments has compromised the functional analysis of viral proteins. In the present study we have performed a functional analysis of the movement proteins (NSM) of four tospovirus species Bean necrotic mosaic virus (BeNMV), Chrysanthemum stem necrosis virus (CSNV), Tomato chlorotic spot virus (TCSV) and Tomato spotted wilt virus (TSWV), which differ biologically and molecularly, by using the Alfalfa mosaic virus (AMV) model system. All NSM proteins were competent to: i) support the cell-to-cell and systemic transport of AMV, ii) generate tubular structures on infected protoplast and iii) transport only virus particles. However, the NSM of BeNMV (one of the most phylogenetically distant species) was very inefficient to support the systemic transport. Deletion assays revealed that the C-terminal region of the BeNMV NSM, but not that of the CSNV, TCSV and TSWV NSM proteins, was dispensable for cell-to-cell transport, and that all the non-functional C-terminal NSM mutants were unable to generate tubular structures. Bimolecular fluorescence complementation analysis revealed that the C-terminus of the BeNMV NSM was not required for the interaction with the cognate nucleocapsid protein, showing a different protein organization when compared with other movement proteins of the '30K family'. Overall, our results revealed clearly differences in functional aspects among movement proteins from divergent tospovirus species that have a distinct biological behavior.

Resistance to Tospoviruses in Vegetable Crops: Epidemiological and Molecular Aspects.

During the past three decades, the economic impact of tospoviruses has increased, causing high yield losses in a variety of crops and ornamentals. Owing to the difficulty in combating thrips vectors with insecticides, the best way to limit/prevent tospovirus-induced diseases involves a management strategy that includes virus resistance. This review briefly presents current tospovirus taxonomy, diversity, molecular biology, and cytopathology as an introduction to a more extensive description of the two main resistance genes employed against tospoviruses: the Sw5 gene in tomato and the Tsw in pepper. Natural and experimental resistance-breaking (RB) isolates allowed the identification of the viral avirulence protein triggering each of the two resistance gene products; epidemiology of RB isolates is discussed to reinforce the need for allelic variants and the need to search for new/alternative resistance genes. Ongoing efforts for alternative resistance strategies are described not only for Tomato spotted wilt virus (TSWV) in pepper and tomato but also for other vegetable crops heavily impacted by tospoviruses.

Biological and molecular characterization of a highly divergent johnsongrass mosaic virus isolate from Pennisetum purpureum.

The complete genome sequence (9,865 nucleotides) of a highly divergent johnsongrass mosaic virus isolate (JGMV-CNPGL) was determined using Illumina sequencing. This isolate infected 10 genotypes of gramineous plants including maize. A comparative analysis of the complete genome showed 80 % nucleotide (nt) sequence identity (86 % amino acid (aa) sequence identity) to a johnsongrass mosaic virus isolate from Australia. The coat protein (CP) identity values, however, were lower than those for the whole genome (78 % and 80 % for nt and aa, respectively) and were close to the species demarcation values (77 % nt and 80 % aa). Unexpectedly, the amino-terminal portion of CP of JGMV-CNPGL showed only 38 % sequence identity to other JGMV isolates. The biological implications of this sequence divergence remain to be elucidated.

Recessive Resistance Derived from Tomato cv. Tyking-Limits Drastically the Spread of Tomato Yellow Leaf Curl Virus.

The tomato yellow leaf curl disease (TYLCD) causes severe damage to tomato (Solanum lycopersicum L.) crops throughout tropical and subtropical regions of the world. TYLCD is associated with a complex of single-stranded circular DNA plant viruses of the genus Begomovirus (family Geminiviridae) transmitted by the whitefy Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae). The tomato inbred line TX 468-RG is a source of monogenic recessive resistance to begomoviruses derived from the hybrid cv. Tyking F1. A detailed analysis of this germplasm source against tomato yellow leaf curl virus-Israel (TYLCV-IL), a widespread TYLCD-associated virus, showed a significant restriction to systemic virus accumulation even under continuous virus supply. The resistance was effective in limiting the onset of TYLCV-IL in tomato, as significantly lower primary spread of the virus occurred in resistant plants. Also, even if a limited number of resistant plants could result infected, they were less efficient virus sources for secondary spread owing to the impaired TYLCV-IL accumulation. Therefore, the incorporation of this resistance into breeding programs might help TYLCD management by drastically limiting TYLCV-IL spread.

The Tomato spotted wilt virus cell-to-cell movement protein (NSM ) triggers a hypersensitive response in Sw-5-containing resistant tomato lines and in Nicotiana benthamiana transformed with the functional Sw-5b resistance gene copy.

Although the Sw-5 gene cluster has been cloned, and Sw-5b has been identified as the functional gene copy that confers resistance to Tomato spotted wilt virus (TSWV), its avirulence (Avr) determinant has not been identified to date. Nicotiana tabacum 'SR1' plants transformed with a copy of the Sw-5b gene are immune without producing a clear visual response on challenge with TSWV, whereas it is shown here that N. benthamiana transformed with Sw-5b gives a rapid and conspicuous hypersensitive response (HR). Using these plants, from all structural and non-structural TSWV proteins tested, the TSWV cell-to-cell movement protein (NSM ) was confirmed as the Avr determinant using a Potato virus X (PVX) replicon or a non-replicative pEAQ-HT expression vector system. HR was induced in Sw-5b-transgenic N. benthamiana as well as in resistant near-isogenic tomato lines after agroinfiltration with a functional cell-to-cell movement protein (NSM ) from a resistance-inducing (RI) TSWV strain (BR-01), but not with NSM from a Sw-5 resistance-breaking (RB) strain (GRAU). This is the first biological demonstration that Sw-5-mediated resistance is triggered by the TSWV NSM cell-to-cell movement protein.

Genetic diversity and recombination analysis of sweepoviruses from Brazil.

BACKGROUND: Monopartite begomoviruses (genus Begomovirus, family Geminiviridae) that infect sweet potato (Ipomoea batatas) around the world are known as sweepoviruses. Because sweet potato plants are vegetatively propagated, the accumulation of viruses can become a major constraint for root production. Mixed infections of sweepovirus species and strains can lead to recombination, which may contribute to the generation of new recombinant sweepoviruses. RESULTS: This study reports the full genome sequence of 34 sweepoviruses sampled from a sweet potato germplasm bank and commercial fields in Brazil. These sequences were compared with others from public nucleotide sequence databases to provide a comprehensive overview of the genetic diversity and patterns of genetic exchange in sweepoviruses isolated from Brazil, as well as to review the classification and nomenclature of sweepoviruses in accordance with the current guidelines proposed by the Geminiviridae Study Group of the International Committee on Taxonomy of Viruses (ICTV). Co-infections and extensive recombination events were identified in Brazilian sweepoviruses. Analysis of the recombination breakpoints detected within the sweepovirus dataset revealed that most recombination events occurred in the intergenic region (IR) and in the middle of the C1 open reading frame (ORF). CONCLUSIONS: The genetic diversity of sweepoviruses was considerably greater than previously described in Brazil. Moreover, recombination analysis revealed that a genomic exchange is responsible for the emergence of sweepovirus species and strains and provided valuable new information for understanding the diversity and evolution of sweepoviruses.

Sequence determination and analysis of the NSs genes of two tospoviruses.

The tospoviruses groundnut ringspot virus (GRSV) and zucchini lethal chlorosis virus (ZLCV) cause severe losses in many crops, especially in solanaceous and cucurbit species. In this study, the non-structural NSs gene and the 5'UTRs of these two biologically distinct tospoviruses were cloned and sequenced. The NSs sequence of GRSV and ZLCV were both 1,404 nucleotides long. Pairwise comparison showed that the NSs amino acid sequence of GRSV shared 69.6% identity with that of ZLCV and 75.9% identity with that of TSWV, while the NSs sequence of ZLCV and TSWV shared 67.9% identity. Phylogenetic analysis based on NSs sequences confirmed that these viruses cluster in the American clade.

A novel monopartite begomovirus infecting sweet potato in Brazil.

The complete genome sequences of two monopartite begomovirus isolates (genus Begomovirus, family Geminiviridae) present in a single sweet potato (Ipomoea batatas) plant collected in São Paulo, Brazil, are presented. Based on the current taxonomic criteria for the genus Begomovirus, one of the isolates was shown to represent a novel species, tentatively named Sweet potato leaf curl Sao Paulo virus (SPLCSPV). The other isolate represented a new strain of sweet potato leaf curl virus, named sweet potato leaf curl virus-Sao Paulo (SPLCV-SP). The full genome sequence of the SPLCSPV isolate shared the highest nucleotide identity (87.6%) with isolates of sweet potato leaf curl Spain virus (SPLCESV). Phylogenetic and recombination analyses were used to investigate the relationships of these isolates to other monopartite Ipomoea-infecting begomoviruses.

Synergistic Interaction Between Tomato chlorosis virus and Tomato spotted wilt virus Results in Breakdown of Resistance in Tomato.

ABSTRACT Multiple viral infections frequently are found in single plants of cultivated and wild hosts in nature, with unpredictable pathological consequences. Synergistic reactions were observed in mixed infections in tomato plants doubly infected with the positive-sense and phloem-limited single-stranded RNA (ssRNA) crinivirus Tomato chlorosis virus (ToCV) and the negative-sense ssRNA tospovirus Tomato spotted wilt virus (TSWV). Synergism in a tomato cultivar susceptible to both viruses resulted in a rapid death of plants. A pronounced enhancement of ToCV accumulation mediated by TSWV co-infection was observed with no evident egress of ToCV from phloem tissues. No consistent alteration of TSWV accumulation was detected. More remarkable was the synergism observed in tomato cultivars which carry the Sw-5 resistance gene, which are resistant to TSWV. Pre-infection with ToCV resulted in susceptibility to TSWV, whereas co-inoculations did not. This suggested that a threshold level or a time lapse is needed for ToCV to interfere or downregulate the defense response in the TSWV-resistant plants.

Erigeron Witches'-Broom Phytoplasma in Brazil Represents New Subgroup VII-B in 16S rRNA Gene Group VII, the Ash Yellows Phytoplasma Group.

A previously undescribed phytoplasma, Erigeron witches'-broom phytoplasma, was detected in diseased plants of Erigeron sp. and Catharanthus roseus exhibiting symptoms of witches'-broom and chlorosis in the state of São Paulo, Brazil. On the basis of restriction fragment length polymorphism (RFLP) analysis of 16S rDNA amplified in the polymerase chain reaction (PCR), Erigeron witches'-broom phytoplasma was classified in group 16SrVII (ash yellows phytoplasma group), new subgroup VII-B. Phylogenetic analysis of 16S rDNA sequences indicated that this phytoplasma represents a new lineage that is distinct from that of described strains of ash yellows phytoplasma. Erigeron witches'-broom phytoplasma is the first member of the ash yellows phytoplasma group to be recorded in Brazil.

Design of a Polymerase Chain Reaction for Specific Detection of Corn Stunt Spiroplasma.

Corn stunt disease is a major limiting factor in production of corn (Zea mays) in the Americas. To develop a polymerase chain reaction (PCR) assay specific for detection of the causal agent, Spiroplasma kunkelii, PCR primers were designed on the basis of unique regions of the nucleotide sequence of the S. kunkelii spiralin gene. DNA was amplified in PCRs containing template DNAs derived from laboratory strains of S. kunkelii and from naturally diseased corn plants collected in the field. No DNA amplification was observed in PCRs containing template DNAs derived from other Spiroplasma species tested or from healthy corn or corn infected by maize bushy stunt phytoplasma. The availability of a sensitive and specific PCR for detection and identification of S. kunkelii should facilitate studies of the ecology of this pathogen, as well as its influence in the incidence, spread, and severity of corn stunting diseases.