Isometric Virus-like Particles Research Articles

Genome Analysis and Pathobiology of Cassava-Infecting Torradoviruses Containing a Putative Maf/HAM1 Pyrophosphatase Domain.

Next generation sequencing has been used to identify and characterize the full genome sequence of a cassava-infecting torradovirus, revealing the presence of a Maf/HAM1 domain downstream of the RNA-dependent RNA-polymerase (RdRp) domain in RNA1 in all isolates sequenced. A similar domain is also found in unrelated potyvirids infecting Euphorbiaceae hosts in the Americas and cassava in Africa. Even though cassava torrado-like virus (CsTLV) could not be mechanically transmitted to a series of herbaceous hosts, it can be efficiently transmitted by bud graft-inoculation to different cassava landraces. Our bioassays show that CsTLV has a narrow host range. Crystal-like structures of isometric virus-like particles were observed in cells of plants with single infection by CsTLV, and consistently induced chlorotic leaf spots and affected root yields significantly. Moreover, CsTLV infection induces changes in the accumulation of total sugars in storage roots. Field surveys indicated the presence of CsTLV in the main cassava growing regions of Colombia, and the occurrence of two different cassava-infecting torradovirus species. Profiles of small RNAs of 21 to 24 nucleotides in length, derived from CsTLV RNAs targeted by cassava RNA silencing defense mechanisms, are also reported.

A novel chrysovirus from a clinical isolate of Aspergillus thermomutatus affects sporulation.

A clinical isolate of Aspergillus thermomutatus (Teleomorph: Neosartorya pseudofischeri) was found to contain ~35 nm isometric virus-like particles associated with four double-stranded (ds) RNA segments, each of which coded for a single open reading frame. The longest dsRNA element (3589 nt) encodes a putative RNA-dependent RNA polymerase (1114 aa), the second longest dsRNA element (2772 nt) encodes a coat protein (825 aa), and the other two dsRNAs (2676 nt, 2514 nt) encode hypothetical proteins of 768 aa and 711 aa, respectively. Phylogenetic analysis of the amino acid sequences showed 41–60% similarity to the proteins coded by the dsRNAs of the most closely related virus, Penicillium janczewskii chrysovirus 2, indicating that it is a new species based on the International Committee on Taxonomy of Viruses criteria for the genus Chrysovirus. This is the first virus reported from A. thermomutatus and was tentatively named Aspergillus thermomutatus chrysovirus 1. A virus free line of the fungal isolate, cured by cycloheximide treatment, produced large numbers of conidia but no ascospores at both 20°C and 37°C, whereas the virus infected line produced ten-fold fewer conidia at 20°C and a large number of ascospores at both temperatures. The effects of the virus on fungal sporulation have interesting implications for the spread of the fungus and possible use of the virus as a biological control agent.

Molecular Characterization of a Trisegmented Mycovirus from the Plant Pathogenic Fungus Colletotrichum gloeosporioides.

A novel double-stranded RNA (dsRNA) mycovirus, consisting of three dsRNA genome segments and possibly belonging to the family Chrysoviridae, was isolated from the filamentous phytopathogenic fungus Colletotrichum gloeosporioides and designated as Colletotrichum gloeosprioides chrysovirus 1 (CgCV1). The three dsRNAs of the CgCV1 genome with lengths of 3397, 2869, and 2630 bp (dsRNAs1–3) were found to contain a single open reading frame (ORF) putatively encoding the RNA-dependent RNA polymerase (RdRp), a capsid protein, and a protease, respectively, all of which exhibited some degree of sequence similarity to the comparable putative proteins encoded by the genus Chrysovirus. The 5′- and 3′-untranslated regions in each dsRNA segment contained similar sequences that were strictly conserved at the termini. Moreover, isometric virus-like particles (VLPs) with a diameter of approximately 40 nm were extracted from fungal mycelia. Phylogenetic analysis based on the conserved dsRNA1-encoded RdRp showed that CgCV1 is a new virus belonging to the Chrysoviridae family. BLAST analysis revealed the presence of CgCV1-like sequences in the chromosomes of Medicago truncatula and Solanum tuberosum. Moreover, some sequences in the transcriptome shotgun assembly (TSA) library and expressed sequence tag database (ESTdb) of other eudicot and monocot plants were also found to be related to CgCV1.

The spectrum of viruses isolated from Pulsatilla pratensis (Ranunculaceae) a native plant of Ukraine

The article is devoted to virus screening of wild plants of Ukraine’s flora. The object of the research is the Red Book plant Pulsatilla pratensis (L.) Mill., which grows on the territory of Kanev Nature Reserve. Isolated isometric infectious virus-like particles with diameters of 34, 36, 43, 47, 50 and 57 nm were isolated from selected plants of P. pratensis. In our research, determination of the infectious nature of the pathogen, host range, concentration of viruses in plants, species identity and virus isolation from the mixture in mixed viral infections were carried with using indicator plants. The typical viral symptoms were observed on indicator plants: browning of the leaf plate, mottling, chlorosis and necrosis. All symptoms were systemic and could be caused by a variety of viruse species. Virions with sizes from 34 to 43 nm produced the necrotic and chlorotic spotting on Chenopodium amaranticolor Coste and Reyn. On the other hand, virions with sizes from 47 to 57 nm produced the necrosis, chlorosis and deformation of the leaf plates on Cucumis sativus L. That is not typical for viruses previously discovered on P. pratensis. The viruses isolated in these plants viruses were cumulated in small concentrations and rapidly lost their infectivity. The number of isolated viruses was insufficient for their identification. Four bacteriophage isolates with long phage tails of different size were isolated from P. pratensis roots and radical soil. The biological (lytic) activity towards the tracer bacteria, the morphology of negative colonies, and bacteriophage protein structure were characterized. According to our research, it is possible to divide phages into three subgroups that probably correspond to three different types of viruses. Results of the polypeptide analysis may reflect an evolutionary process in a population of phages that had a common ancestor. Comparison of phage proteins of different hosts shows a variety of molecular weights of polypeptides comprising up to several dozens of proteins. The results of the current work lay the basis for studying the spread of viruses in nature and determination of their relationships in biocenoses.

Molecular characterization of a novel victorivirus from the entomopathogenic fungus Beauveria bassiana

New Zealand isolates of the entomopathogenic fungus Beauveria were examined for the presence of dsRNAs and virus-like particles. Seven out of nine isolates contained one or more high-molecular-weight dsRNAs and all seven contained isometric virus particles ranging in size from 30 to 50 nm. B. bassiana isolate ICMP#6887 contained a single dsRNA band of ~6 kb and isometric virus-like particles of ~50 nm in diameter. Sequencing revealed that the virus from ICMP#6887 had a genome of 5,327 nt with two overlapping ORFs coding for a putative coat protein (CP) and an RNA-dependent RNA-polymerase (RdRp). The sequence showed a highest CP identity of 58.3 % to Tolypocladium cylindrosporum virus 1 (TcV1) and a highest RdRp identity of 48.8 % to Sphaeropsis sapinea RNA virus 1 (SsRV1). Since both TcV1 and SsRV1 belong to the genus Victorivirus, the new virus from B. bassiana ICMP#6887 was tentatively assigned the name Beauveria bassiana victorivirus 1 (BbVV1-6887).

Molecular detection of an isolate of Cucumber mosaic virus infecting Oxalis corymbosa in Aligarh, India

Plants of Oxalis corymbosa displaying typical viral symptoms were observed during spring 2010 in plant nurseries in central India. The virus was mechanically transmissible to indicator plants and induced symptoms similar to those incited by Cucumber mosaic virus (CMV). Electron microscopic examinations of symptomatic leaf samples revealed the presence of isometric virus-like particles about 28–30 nm in diameter. Molecular detection methods allowed the identification of the pathogen as a CMV. The results were confirmed by reverse transcription polymerase chain reaction with specific CMV primers, and digestion with a restriction enzyme. The coat protein gene was 95 and 99% identical at the nucleotide and amino acid levels, respectively, to the published sequences of CMV, high amino acid sequence homology in multiple alignment analysis and closest phylogenetic relationship with Lucknow isolate (JX570732) of CMV. This represents the first record for CMV-infecting O. corymbosa in Aligarh India.

Molecular characterization of a trisegmented chrysovirus isolated from the radish Raphanus sativus

Radish (Raphanus sativus L.) is cultivated worldwide and is of agronomic importance. dsRNAs associated with partitiviruses were previously found in many R. sativus varieties. In this study, three large dsRNAs from radish were cloned using a modified single primer amplification technique. These three dsRNAs—of lengths 3638, 3517 and 3299 bp—shared conserved untranslated terminal regions, and each contained a major open reading frame putatively encoding the chrysoviral replicase, capsid protein and protease respectively. Isometric virus-like particles (VLP), approximately 45nm in diameter, were isolated from the infected radish plants. Northern blotting indicated that these dsRNAs were encapsidated in the VLP. The virus containing these dsRNA genome segments was named Raphanus sativus chrysovirus 1 (RasCV1). Phylogenetic analysis revealed that RasCV1 is a new species of the Chrysoviridae family and forms a plant taxon with another putative plant chrysovirus, Anthurium mosaic-associated virus (AmaCV). Furthermore, no fungal mycelia were observed in radish leaf tissues stained with trypan blue. These results indicated that RasCV1 is most likely a plant chrysovirus rather than a chrysovirus in symbiotic fungi. An exhaustive BLAST analysis of RasCV1 and AmaCV revealed that chrysovirus-like viruses might widely exist in eudicot and monocot plants and that endogenization of chrysovirus segments into plant genome might have ever happened.

Genomic characterization of a novel dsRNA virus detected in the phytopathogenic fungus Verticillium dahliae Kleb.

Four novel double-stranded RNA segments were detected in a Verticillium dahliae Kleb. strain ( V. dahliae isolate 0-21), a causal fungal agent of Verticillium wilt disease of cotton. Each dsRNA genome segment contains a single large open reading frame (ORF) that encodes a distinctive protein with modest levels of sequence similarities to the corresponding putative proteins in the genus Chrysovirus. These include an RNA-dependent RNA polymerase (RdRp), a coat protein, an undefined replication-related protein and an ovarian tumor domain peptidase. Phylogenetic analysis of the four putative proteins unanimously indicated that they are evolutionarily related to viruses in Chrysovirus. The 5′- and 3′-untranslated regions of the four dsRNAs share highly similar internal sequence and contain conserved sequence stretches of UGAUAAAAAA(/U)UG(/U)AAAAA- (in the 5′-UTR) and -UUUACUACU (in the 3′-UTR), indicating that they have a common virus origin. Indeed, isometric virus-like particles (VLPs) with a diameter of approximately 34 nm were extracted from the fungal mycelia, and the four dsRNA segments were also detected in the virus-like particle (VLP) fraction. These results suggest that the mycovirus with four different dsRNA genome segments from the fungal isolate 0-21 is a new member of the genus Chrysovirus. We named the virus Verticillium dahliae chrysovirus 1 (VdCV1).

국내 복숭아에서 분리한 Prunus necrotic ringspot virus의 특성

In this paper, we report a characterization of Prunus necrotic ringspot virus (PNRSV) isolate. The virus was identified from `Yumyeong` peach showing mild mosaic on leaves in commercial orchard of `Umsung`, Chungbuk province in Korea. The virus isolate produced ringspot symptom on the inoculated cotyledons and systemic mosaic and malformation on the upper leaves of Cucumis sativus. Systemic mottles were appeared in Chenopodium quinoa. When the buds of the virus infected stem were grafted on the healthy young Prunus persica GF305 seedlings, line pattern with mosaic appeared within 3 months. Isometric virus-like particles were found in parenchyma cells and plasmodesmata of C. sativus leaves inoculated mechanically with the virus. The cDNA fragments of PNRSV coat protein (CP) region, approximately 675bp, were synthesized from genomic RNA extracted from virus-infected leaves by RT-PCR using specific primer pairs. Partial nucleotide sequences of the CP regions were determined and analyzed with the known PNRSV. The CP gene of PNRSVKorea isolates showed 93.9~94.7% similarity to the 4 known PNRSV isolates.

Putative agents of fig mosaic disease in Turkey

Leaves from 58 fig tree accessions showing different severity of mosaic and necrotic symptoms were collected in 2 different regions of Turkey and analysed by transmission electron microscope (TEM) and RT-PCR to determine putative causal agents of fig mosaic disease (FMD). Ultrastructural studies showed that double membrane-bound bodies (DMBs) were mainly observed in the parenchyma and mesophyll cells of infected leaves of the cultivars Göklop and Morgüz and some seedlings showing only mosaic symptoms. These bodies were surrounded by a fibrillar matrix in most of the infected cells. Long, flexuous rod-shaped virus-like particles (LFPs) were also observed in vascular tissues of the Bursa Siyahı, Sarılop, Sarızeybek, and Yediveren cultivars, as well as seedlings that exhibited mosaic and necrotic symptoms together. Isometric virus-like particles (IVLPs) were mainly observed in mesophyll parenchyma cells of cv. Yeşilgüz, which showed very mild mosaic symptoms. Some phytoplasma-like bodies, as well as mosaic symptoms, were also observed in the phloem tissues of cv. Morgüz, Göklop, and Yediveren. These kinds of particles were not observed in the healthy plant leaves. RT-PCR analyses revealed that a PCR product of 3.0 kbp was obtained from virus-purified RNA of cvs. Bursa Siyahı, and Sarılop and an unknown cultivar (seedling) using degenerate fig-associated primers. When primers specific for FMD agent in Spain were used, a DNA fragment of 750 bp was amplified from cvs. Göklop and Yeşilgüz. However, no product was obtained by using DC-random primers.

Exploiting the Yeast L-A Viral Capsid for the In Vivo Assembly of Chimeric VLPs as Platform in Vaccine Development and Foreign Protein Expression

A novel expression system based on engineered variants of the yeast (Saccharomyces cerevisiae) dsRNA virus L-A was developed allowing the in vivo assembly of chimeric virus-like particles (VLPs) as a unique platform for a wide range of applications. We show that polypeptides fused to the viral capsid protein Gag self-assemble into isometric VLP chimeras carrying their cargo inside the capsid, thereby not only effectively preventing proteolytic degradation in the host cell cytosol, but also allowing the expression of a per se cytotoxic protein. Carboxyterminal extension of Gag by T cell epitopes from human cytomegalovirus pp65 resulted in the formation of hybrid VLPs that strongly activated antigen-specific CD8+ memory T cells ex vivo. Besides being a carrier for polypeptides inducing antigen-specific immune responses in vivo, VLP chimeras were also shown to be effective in the expression and purification of (i) a heterologous model protein (GFP), (ii) a per se toxic protein (K28 α-subunit), and (iii) a particle-associated and fully recyclable biotechnologically relevant enzyme (esterase A). Thus, yeast viral Gag represents a unique platform for the in vivo assembly of chimeric VLPs, equally attractive and useful in vaccine development and recombinant protein production.

A double-stranded RNA mycovirus in Botrytis cinerea.

In wild-type Botrytis cinerea CVg25 strain we have detected the presence of extrachromosomal genetic elements corresponding to double-stranded RNA molecules. These genetic elements have been designated L, M1 and M2 with molecular sizes of 8.3, 2.0 and 1.4 kb, respectively. The visualization by electron microscopy of mycelium ultrathin sections from B. cinerea CVg25 showed the presence of isometric virus-like particles of about 40 nm in diameter. Linear sucrose gradient centrifugation of mycelium-free extracts was done to determine if the double-stranded RNAs were associated with virus-like particles. The gradient profile obtained at 260 and 280 nm revealed a major peak that was analyzed by both agarose-gel electrophoresis and electron microscopy. It was observed that only the L-double-stranded RNA molecule copurified with isometric virus-like particles. These virus-like particles had a similar morphology and size as those detected by electron microscopy in the mycelium sections. These results suggest that only the L-double-stranded RNA would be encapsidated.

Brief Note: Natural infection of Viola cornuta (Violaceae) with Cucumber mosaic virus, subgroup I

Plants of Viola cornuta displaying typical virus symptoms were observed during spring 2003 in a plant nursery in Córdoba, central Argentina. Electron microscopic examinations of symptomatic leaf samples revealed the presence of isometric virus-like particles about 30 nm in diameter. Subsequent serological analysis allowed the identification of the pathogen as a subgroup I strain of Cucumber mosaic virus (CMV). These results were confirmed by antigen capture--reverse transcription--polymerase chain reaction with specific CMV primers, and digestion with a restriction enzyme. This is the first report of CMV infecting V. cornuta in Argentina.

Aphid Acquisition and Cellular Transport of Potato leafroll virus-like Particles Lacking P5 Readthrough Protein

ABSTRACT Lepidopteran cells (Spodoptera frugiperda) produced isometric virus-like particles (VLP) when infected with a recombinant baculovirus Ac61 that contained the Potato leafroll virus (PLRV) coat protein gene modified with an N-terminal histidine tag (P3-6H). Cells infected with AcFL, a recombinant baculovirus that expressed cDNA copies of the PLRV genome RNA, did not produce virus-like particles (VLP). In cell lines doubly infected with Ac61 and AcFL, VLP were formed that contained PLRV-RNA packaged in P3-6H coat protein (FL). Both the P3-6H and the FL particles were morphologically indistinguishable from particles of PLRV despite the fact that they lacked the P5 readthrough protein present in wild-type PLRV. When aphids (Myzus persicae) were fed on, or injected with, purified PLRV, or VLP of either type (FL or P3-6H) and examined by electron microscopy, no differences were observed among treatments for particle endocytosis, transcellular transport, or exocytosis at the aphid midgut or accessory salivary glands. Particles were observed in the salivary canals and in the salivary duct leading out of the aphid. These results suggest that P5 readthrough protein of PLRV may not be essential for cellular transport of virus through aphid vectors.

Double-stranded RNA and virus-like particles in the grass endophyte Epichloë festucae

Two double-stranded ribonucleic acid (dsRNA) elements were detected in an isolate of the clavicipitaceous grass endophyte Epichloe festucae obtained from an asymptomatic Festuca rubra plant. No dsRNA was detected in three other isolates of E. festucae. The size of the two dsRNA elements isolated from mycelium was of 5.2 and 3.2 kbp. Hybridization experiments with a cDNA probe complementary to the 5.2 kbp dsRNA showed that there was no sequence similarity between the dsRNA elements. Isometric virus-like particles of about 50 nm in diameter were observed by electron microscopy in sucrose-gradient purified virus preparations from the isolate containing dsRNA. The 5.2 kbp dsRNA element was present in the partially purified virus preparations, suggesting that this element is encapsidated. There was no difference with respect to growth rate or colony morphology between the dsRNA containing and dsRNA-free isolates. The presence of dsRNA and virus-like particles suggests the presence of a mycovirus in the Vit5 strain of E. festucae.

A double-stranded RNA mycovirus in Botrytis cinerea

In wild-type Botrytis cinerea CVg25 strain we have detected the presence of extrachromosomal genetic elements corresponding to double-stranded RNA molecules. These genetic elements have been designated L, M 1 and M 2 with molecular sizes of 8.3, 2.0 and 1.4 kb, respectively. The visualization by electron microscopy of mycelium ultrathin sections from B. cinerea CVg25 showed the presence of isometric virus-like particles of about 40 nm in diameter. Linear sucrose gradient centrifugation of mycelium-free extracts was done to determine if the double-stranded RNAs were associated with virus-like particles. The gradient profile obtained at 260 and 280 nm revealed a major peak that was analyzed by both agarose-gel electrophoresis and electron microscopy. It was observed that only the L-double-stranded RNA molecule copurified with isometric virus-like particles. These virus-like particles had a similar morphology and size as those detected by electron microscopy in the mycelium sections. These results suggest that only the L-double-stranded RNA would be encapsidated.

Identification of Piper yellow mottle virus, a mealybug-transmitted badnavirus infecting Piper spp. in Southeast Asia

A previously undescribed badnavirus was found to be a causal agent of a disease of black pepper (Piper nigrum) in Malaysia, the Philippines, Sri Lanka and Thailand, and was also associated with a disease of betelvine (P. betle) in Thailand. Disease symptoms included chlorotic mottling, chlorosis, vein-clearing, leaf distortion, reduced plant vigor and poor fruit set. The virus, named Piper yellow mottle virus (PYMV), had non-enveloped bacilliform virions averaging 30 × 125 nm in size and containing a double-stranded DNA genome. An isolate of PYMV from Thailand was transmitted by mechanical inoculation and by the citrus mealybug, Planococcus citri, from infected P. nigrum and P. betle to healthy P. nigrum seedlings, which developed symptoms similar to those observed in naturally-infected plants. A serological relationship between PYMV and isolates of banana streak (BSV) and sugarcane bacilliform (ScBV) viruses, but not six other badnaviruses, was detected by immunosorbent electron microscopy (ISEM). Genomic PYMV sequences were amplified by polymerase chain reaction (PCR) using badnavirus-specific oligonucleotide primers, and sequence analysis comparisons of the putative reverse transcriptase (RT) domain showed PYMV to be closely related to other mealybug-transmitted badnaviruses. Black pepper infected with PYMV sometimes contained one or more isometric virus-like particles, and PYMV may therefore be only one component of a virus complex infecting black pepper in Southeast Asia.

Sugarcane Yellow Leaf Disease in Brazil: Evidence of Association with a Luteovirus.

Evidence of the viral etiology of sugarcane yellow leaf disease (SCYLD), occurring in southeast Brazil, was obtained by light and electron microscopy combined with serology. Light microscopy using epifluorescence illumination showed an abnormal yellow-green fluorescing material in the phloem of SCYLD-affected plants that was rarely observed in control plants. Immunolocalization in tissue-printed (or -blotted) nitrocellulose membranes, using barley yellow dwarf virus (BYDV) serotype PAV antiserum, showed a weak but clearly positive reaction in the phloem. Isometric viruslike particles of 24 to 26 nm in diameter were found by electron microscopy both in situ and in partially purified preparations. Examination of thin sections showed that phloem companion cells contained viruslike particles and presented cytological changes apparently related to the development of virus infection. Partially purified preparations produced UV absorption spectra typical of a nucleoprotein, with high absorbance at 260 nm, as expected for isometric virus particles. Virus particles were observed in extracts and partially purified preparations using immunosorbent electron microscopy with BYDV-PAV antiserum. Plate-trapped antigen enzyme-linked immunosorbent assays with the same antiserum indicate a weak serological relationship between BYDV-PAV and SCYLD-associated virus.

Double-stranded RNA and isometric virus-like particles in the entomopathogenic fungus Metarhizium anisopliae

Seven wild-type isolates of the entomopathogenic Metashizium anisopliae were analysed for the presence of a mycoviral infection. Extra bands of dsRNA were detected in three of them via agarose gel electrophoresis. Strain RJ has bands of dsRNA varying from 910 to 3100 bp. One associated protein (46 kDa) was identified. The presence of virus-like particles of ca 35 nm of diameter was confirmed by TEM. The pattern of dsRNA was verified in both mycelium and spores. Auxotrophic mutants derived from strain RJ exhibited the same dsRNA band pattern and similar virus-like particles.

Purification and characterization of a virus-like particle ofLeucostoma persoonii

Abundant isometric virus-like particles were visible in hyphae of a hypovirulent isolate of Leucostoma persoonii, NC14.4A, that also contained at least six segments of double-stranded RNA. We purif...