Aedes aegypti Aag-2 Cell Proteome Modulation in Response to Chikungunya Virus Infection.

Chikungunya virus (CHIKV) is a single-stranded positive RNA virus that belongs to the genus Alphavirus and is transmitted to humans by infected Aedes aegypti and Aedes albopictus bites. In humans, CHIKV usually causes painful symptoms during acute and chronic stages of infection. Conversely, virus-vector interaction does not disturb the mosquito's fitness, allowing a persistent infection. Herein, we studied CHIKV infection of Ae. aegypti Aag-2 cells (multiplicity of infection (MOI) of 0.1) for 48 h through label-free quantitative proteomic analysis and transmission electron microscopy (TEM). TEM images showed a high load of intracellular viral cargo at 48 h postinfection (hpi), as well as an unusual elongated mitochondria morphology that might indicate a mitochondrial imbalance. Proteome analysis revealed 196 regulated protein groups upon infection, which are related to protein synthesis, energy metabolism, signaling pathways, and apoptosis. These Aag-2 proteins regulated during CHIKV infection might have roles in antiviral and/or proviral mechanisms and the balance between viral propagation and the survival of host cells, possibly leading to the persistent infection.

In silico construction of a multiepitope Zika virus vaccine using immunoinformatics tools.

Zika virus (ZIKV) is an arbovirus from the Flaviviridae family and Flavivirus genus. Neurological events have been associated with ZIKV-infected individuals, such as Guillain-Barré syndrome, an autoimmune acute neuropathy that causes nerve demyelination and can induce paralysis. With the increase of ZIKV infection incidence in 2015, malformation and microcephaly cases in newborns have grown considerably, which suggested congenital transmission. Therefore, the development of an effective vaccine against ZIKV became an urgent need. Live attenuated vaccines present some theoretical risks for administration in pregnant women. Thus, we developed an in silico multiepitope vaccine against ZIKV. All structural and non-structural proteins were investigated using immunoinformatics tools designed for the prediction of CD4 + and CD8 + T cell epitopes. We selected 13 CD8 + and 12 CD4 + T cell epitopes considering parameters such as binding affinity to HLA class I and II molecules, promiscuity based on the number of different HLA alleles that bind to the epitopes, and immunogenicity. ZIKV Envelope protein domain III (EDIII) was added to the vaccine construct, creating a hybrid protein domain-multiepitope vaccine. Three high scoring continuous and two discontinuous B cell epitopes were found in EDIII. Aiming to increase the candidate vaccine antigenicity even further, we tested secondary and tertiary structures and physicochemical parameters of the vaccine conjugated to four different protein adjuvants: flagellin, 50S ribosomal protein L7/L12, heparin-binding hemagglutinin, or RS09 synthetic peptide. The addition of the flagellin adjuvant increased the vaccine's predicted antigenicity. In silico predictions revealed that the protein is a probable antigen, non-allergenic and predicted to be stable. The vaccine's average population coverage is estimated to be 87.86%, which indicates it can be administered worldwide. Peripheral Blood Mononuclear Cells (PBMC) of individuals with previous ZIKV infection were tested for cytokine production in response to the pool of CD4 and CD8 ZIKV peptide selected. CD4 + and CD8 + T cells showed significant production of IFN-γ upon stimulation and IL-2 production was also detected by CD8 + T cells, which indicated the potential of our peptides to be recognized by specific T cells and induce immune response. In conclusion, we developed an in silico universal vaccine predicted to induce broad and high-coverage cellular and humoral immune responses against ZIKV, which can be a good candidate for posterior in vivo validation.

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.

Two viruses from Stylosanthes guianensis may represent a new genus within Potyviridae.

Forage crops occupy large areas of tropical pastures for cattle feeding in Brazil. The use of stylos (Stylosanthes spp.) in these pastures, which are leguminous shrubs, has increased in the country due to their outstanding nutritional value and for being an efficient and alternative source for nitrogen fixation in the soil. In recent years, virus-like mosaic symptoms on S.guianensis leaves have often been observed in the field, indicating possible virus-like pathogen infections. In an effort to identify the causal agent, virus semi-purification protocol was performed using symptomatic S. guianensis leaves collected at EMBRAPA Beef Cattle Research Center. Total RNA extracted from this semi-purified preparation was submitted to high-throughput sequencing, which revealed complete genome sequences of novel viruses of the family Potyviridae. These viruses, tentatively named stylo mosaic-associated virus 1 (StyMaV-1) and stylo mosaic-associated virus 2 (StyMaV-2), shared 73 % CP aa identity and 77 % polyprotein aa identity with each other and, after that, being closest related to blackberry virus Y, genus Brambyvirus (only 41 % CP aa identity). Based on ICTV genus demarcation criteria, StyMaV-1 and StyMaV-2 represent new species of a new genus within the family Potyviridae. StyMaV-1 and StyMaV-2 are also not efficiently transmitted to other plant species by mechanical inoculation.

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.

Dynamic proteomic analysis of Aedes aegypti Aag-2 cells infected with Mayaro virus.

BACKGROUND: Mayaro virus (MAYV) is responsible for a mosquito-borne tropical disease with clinical symptoms similar to dengue or chikungunya virus fevers. In addition to the recent territorial expansion of MAYV, this virus may be responsible for an increasing number of outbreaks. Currently, no vaccine is available. Aedes aegypti is promiscuous in its viral transmission and thus an interesting model to understand MAYV-vector interactions. While the life-cycle of MAYV is known, the mechanisms by which this arbovirus affects mosquito host cells are not clearly understood. METHODS: After defining the best conditions for cell culture harvesting using the highest virus titer, Ae. aegypti Aag-2 cells were infected with a Brazilian MAYV isolate at a MOI of 1 in order to perform a comparative proteomic analysis of MAYV-infected Aag-2 cells by using a label-free semi-quantitative bottom-up proteomic analysis. Time-course analyses were performed at 12 and 48 h post-infection (hpi). After spectrum alignment between the triplicates of each time point and changes of the relative abundance level calculation, the identified proteins were annotated and using Gene Ontology database and protein pathways were annotated using the Kyoto Encyclopedia of Genes and Genomes. RESULTS: After three reproducible biological replicates, the total proteome analysis allowed for the identification of 5330 peptides and the mapping of 459, 376 and 251 protein groups, at time 0, 12 hpi and 48 hpi, respectively. A total of 161 mosquito proteins were found to be differentially abundant during the time-course, mostly related to host cell processes, including redox metabolism, translation, energy metabolism, and host cell defense. MAYV infection also increased host protein expression implicated in viral replication. CONCLUSIONS: To our knowledge, this first proteomic time-course analysis of MAYV-infected mosquito cells sheds light on the molecular basis of the viral infection process and host cell response during the first 48 hpi. Our data highlight several mosquito proteins modulated by the virus, revealing that MAYV manipulates mosquito cell metabolism for its propagation.

An isolate of sweet potato chlorotic stunt virus from Brazil with a distinct genome organization.

Sweet potato chlorotic stunt virus (SPCSV; genus Crinivirus, family Closteroviridae), is an economically important pathogen of sweet potato. In the present work, the nucleotide sequences of two RNA segments of SPCSV (isolate SPCSV-UNB-01) were determined by MiSeq Illumina sequencing of samples of sweet potato plants grafted onto Ipomoea setosa. A comparative analysis of the genome organization of SPCSV-UNB-01 and other SPCSV sequences showed that RNA1 was lacking p22, and p5.1 and that p5.2. was absent in RNA2, indicating a unique genomic pattern. SPCSV-UNB-01 contained longer p6 and p5 regions, with little similarity to orthologous sequences. Sequence comparison did not reveal any previously identified functional domains within these open reading frames (ORFs). No recombination or rearrangement events were detected. Phylogenetic analysis suggested the possibility of separate entries of SPCSV into South America based on the genetic distance between SPCSV-UNB-01 and the Peruvian isolate m2-47. Samples from northeastern Brazil (State of Pernambuco) were positive for SPCSV when tested using specific primers for the major coat protein (CP) gene. This is the first full-length genome sequence of SPCSV-UNB-01 from Brazil.

Tomato Chlorotic Spot Virus (TCSV) Putatively Incorporated a Genomic Segment of Groundnut Ringspot Virus (GRSV) Upon a Reassortment Event.

Tomato chlorotic spot virus (TCSV) and groundnut ringspot virus (GRSV) share several genetic and biological traits. Both of them belong to the genus Tospovirus (family Peribunyaviridae), which is composed by viruses with tripartite RNA genome that infect plants and are transmitted by thrips (order Thysanoptera). Previous studies have suggested several reassortment events between these two viruses, and some speculated that they may share one of their genomic segments. To better understand the intimate evolutionary history of these two viruses, we sequenced the genomes of the first TCSV and GRSV isolates ever reported. Our analyses show that TCSV and GRSV isolates indeed share one of their genomic segments, suggesting that one of those viruses may have emerged upon a reassortment event. Based on a series of phylogenetic and nucleotide diversity analyses, we conclude that the parental genotype of the M segment of TCSV was either eliminated due to a reassortment with GRSV or it still remains to be identified.

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.

Dissecting the Subcellular Localization, Intracellular Trafficking, Interactions, Membrane Association, and Topology of Citrus Leprosis Virus C Proteins.

Citrus leprosis (CL) is a re-emergent viral disease affecting citrus crops in the Americas, and citrus leprosis virus C (CiLV-C), belonging to the genus Cilevirus, is the main pathogen responsible for the disease. Despite the economic importance of CL to the citrus industry, very little is known about the performance of viral proteins. Here, we present a robust in vivo study around functionality of p29, p15, p61, MP, and p24 CiLV-C proteins in the host cells. The intracellular sub-localization of all those viral proteins in plant cells are shown, and their co-localization with the endoplasmic reticulum (ER), Golgi complex (GC) (p15, MP, p61 and p24), actin filaments (p29, p15 and p24), nucleus (p15), and plasmodesmata (MP) are described. Several features are disclosed, including i) ER remodeling and redistribution of GC apparatus, ii) trafficking of the p29 and MP along the ER network system, iii) self-interaction of the p29, p15, and p24 and hetero-association between p29-p15, p29-MP, p29-p24, and p15-MP proteins in vivo. We also showed that all proteins are associated with biological membranes; whilst p15 is peripherally associated, p29, p24, and MP are integrally bound to cell membranes. Furthermore, while p24 exposes an N-cytoplasm-C-lumen topology, p29, and p15 are oriented toward the cytoplasmic face of the biological membrane. Based on our findings, we discuss the possible performance of each protein in the context of infection and a hypothetical model encompassing the virus spread and sites for replication and particle assembly is suggested.

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.

Identification and genome analysis of tomato chlorotic spot virus and dsRNA viruses from coinfected vegetables in the Dominican Republic by high-throughput sequencing.

The Tomato chlorotic spot virus (TCSV) was first reported in the 1980s, having its occurrence limited to Brazil and Argentina. Due to an apparent mild severity in the past, molecular studies concerning TCSV were neglected. However, TCSV has disseminated over the USA and Caribbean countries. In Dominican Republic TCSV has been recently reported on important cultivated crops such as pepper and beans. In this work, we provide the first complete genome of a TCSV isolate from symptomatic plants in Dominican Republic, which was obtained by high-throughput sequencing. In addition, three dsRNA viruses from different virus families were identified coinfecting these plants Bell pepper endornavirus (BPEV), Southern tomato virus (STV) and Pepper cryptic virus 2 (PCV-2). Phylogenetic analysis showed that the Dominican Republic TCSV isolate has a close relationship with other TCSV isolates and a reassortant isolate between TCSV and Groundnut ringspot virus (GRSV), all found in USA. BPEV, STV and PCV-2 isolates from Dominican Republic were close related to corresponding American isolates. The possible biological implications of these virus-mixed infections are discussed.

The NSm proteins of phylogenetically related tospoviruses trigger Sw-5b-mediated resistance dissociated of their cell-to-cell movement function.

The cell-to-cell movement protein (NSM) of tomato spotted wilt virus (TSWV) has been recently identified as the effector of the single dominant Sw-5b resistance gene from tomato (Solanum lycopersicum L.). Although most TSWV isolates shows a resistance-inducing (RI) phenotype, regular reports have appeared on the emergence of resistance-breaking (RB) isolates in tomato fields, and suggested a strong association with two point mutations (C118Y and T120N) in the NSM protein. In this study the Sw-5b gene has been demonstrated to confer not only resistance against TSWV but to members of five additional, phylogenetically-related classified within the so-called "American" evolutionary clade, i.e., Alstroemeria necrotic streak virus (ANSV), chrysanthemum stem necrosis virus (CSNV), groundnut ringspot virus (GRSV), Impatiens necrotic spot virus (INSV) and tomato chlorotic spot virus (TCSV). Remarkably, bean necrotic mosaic virus (BeNMV), a recently discovered tospovirus classified in a distinct American subclade and circulating on the American continent, did not trigger a Sw-5b-mediated hypersensitive (HR) response. Introduction of point mutations C118Y and T120N into the NSM protein of TSWV, TCSV and CSNV abrogated the ability to trigger Sw-5b-mediated HR in both transgenic-N. benthamiana and tomato isolines harboring the Sw-5b gene whereas it had no effect on BeNMV NSM. Truncated versions of TSWV NSM lacking motifs associated with tubule formation, cell-to-cell or systemic viral movement were made and tested for triggering of resistance. HR was still observed with truncated NSM proteins lacking 50 amino acids (out of 301) from either the amino- or carboxy-terminal end. These data altogether indicate the importance of amino acid residues C118 and T120 in Sw-5b-mediated HR only for the NSM proteins from one cluster of tospoviruses within the American clade, and that the ability to support viral cell-to-cell movement is not required for effector functionality.

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.

Host-specific accumulation and temperature effects on the generation of dimeric viral RNA species derived from the S-RNA of members of the Tospovirus genus.

Polygonum ringspot virus (PolRSV) is a recently characterized Tospovirus reported in Italy. Northern blot analyses of PolRSV infections in Nicotiana benthamiana and tomato plants showed that a viral RNA species with nearly twice the length of the Small genomic RNA (S-RNA) accumulated abundantly in the former host, but was not detected in the latter. Additional assays confirmed that biogenesis of this novel RNA species was common to all PolRSV isolates tested and also to an isolate of Tomato spotted wilt virus (TSWV). Given its size, we hypothesized that the novel RNA species was a dimer molecule and we confirmed this hypothesis by RNA sequencing (RNAseq) analysis and reverse transcription (RT)-PCR of putative predicted dimer junction sites in RNA extracts of N. benthamiana challenged with PolRSV isolates Plg6 and Plg13/2. We also confirmed that these molecules are derived from head-to-tail dimers and often contain deletions at their junction sites. We named these novel molecules imperfect dimer RNAs (IMPD-RNAs). PolRSV IMPD-RNAs systemic accumulation in a range of host plants was restricted to N. benthamiana and Nicotiana occidentalis. Notably, IMPD-RNAs accumulation was modulated by temperature and their generation was restricted to late stages of systemic infection (12 days post-inoculation) in N. benthamiana. Differently from all other PolRSV isolates used in this study, Plg13/2 generated more IMPD-RNAs coupled with low amounts of genomic S-RNA and maintained them even at 18 °C, besides having lost the ability to infect tomato plants. This is the first characterization of S-RNA dimers for Tospovirus, and of occurrence of dimers of genomic segments at the whole organism level for Bunyaviridae.

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.