Filamentous Virus Research Articles

Revisiting the cysteine-rich proteins encoded in the 3'-proximal open reading frame of the positive-sense single-stranded RNA of some monopartite filamentous plant viruses: functional dissection of p15 from grapevine virus B.

A reexamination of proteins with conserved cysteines and basic amino acids encoded by the 3'-proximal gene of the positive-sense single-stranded RNA of some monopartite filamentous plant viruses has been carried out. The cysteines are involved in a putative Zn-finger domain, which, together with the basic amino acids, form part of the nuclear or nucleolar localization signals. An in-depth study of one of these proteins, p15 from grapevine B virus (GVB), has shown: (i) a three-dimensional structure with four α-helices predicted by two independent in silico approaches, (ii) the nucleolus as the main accumulation site by applying confocal laser microscopy to a fusion between p15 and the green fluorescent protein, (iii) the involvement of the basic amino acids and the putative Zn-finger domain, mapping at the N-terminal region of p15, in the nucleolar localization signal, as revealed by the effect of six alanine substitution mutations, (iv) the p15 suppressor function of sense-mediated RNA silencing as revealed by agroinfiltration in a transgenic line of Nicotiana benthamiana, and (v) the enhancer activity of p15 on viral pathogenicity in N. benthamiana when expressed from a potato virus X vector. In addition, we elaborate on an evolutionary scenario for these filamentous viruses, invoking takeover by a common ancestor(s) of viral or host genes coding for those cysteine-rich proteins, followed by divergence, which would also explain why they are encoded in the 3'-proximal gene of the genomic single-stranded viral RNA.

ICTV Virus Taxonomy Profile: Alphaflexiviridae.

The family Alphaflexiviridae includes viruses with flexuous filamentous virions that are 470–800 nm in length and 12–13 nm in diameter. Alphaflexiviruses have a single-stranded, positive-sense RNA genome of 5.5–9 kb. They infect plants and plant-infecting fungi. They share a distinct lineage of alphavirus-like replication proteins that is unusual in lacking any recognized protease domain. With a single exception, cell-to-cell and long-distance movement is facilitated by triple gene block proteins in plant-infecting genera. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Alphaflexiviridae, which is available at www.ictv.global/report/alphaflexiviridae.

Shotgun sequencing of honey DNA can describe honey bee derived environmental signatures and the honey bee hologenome complexity

Honey bees are large-scale monitoring tools due to their extensive environmental exploration. In their activities and from the hive ecosystem complex, they get in close contact with many organisms whose traces can be transferred into the honey, which can represent an interesting reservoir of environmental DNA (eDNA) signatures and information useful to analyse the honey bee hologenome complexity. In this study, we tested a deep shotgun sequencing approach of honey DNA coupled with a specifically adapted bioinformatic pipeline. This methodology was applied to a few honey samples pointing out DNA sequences from 191 organisms spanning different kingdoms or phyla (viruses, bacteria, plants, fungi, protozoans, arthropods, mammals). Bacteria included the largest number of species. These multi-kingdom signatures listed common hive and honey bee gut microorganisms, honey bee pathogens, parasites and pests, which resembled a complex interplay that might provide a general picture of the honey bee pathosphere. Based on the Apis mellifera filamentous virus genome diversity (the most abundant detected DNA source) we obtained information that could define the origin of the honey at the apiary level. Mining Apis mellifera sequences made it possible to identify the honey bee subspecies both at the mitochondrial and nuclear genome levels.

Site-specific dynamics of methyl groups probed by temporal evolution of heteronuclear 13C{1H} Overhauser polarisation encoded in 13C – 13C solid-state NMR correlation experiments

There is great interest in studying the dynamics of methyl groups which are sensitive probes of local packing and molecular motions in biosolids and proteins. In this work we show that the magnetisation created by transient heteronuclear 13C{1H} Overhauser effect can be exploited in two-dimensional 13C – 13C solid-state NMR correlation experiments for straightforward access to site-specific dynamics of methyl groups in fully protonated 13C labelled biomolecules. The methodology developed for this purpose is demonstrated on polycrystalline L-isoleucine and applied to probe residue-specific dynamics of methyl groups in the coat protein of Pf1 bacteriophage filamentous virus.

Parasites and pathogens associated with native bumble bees (Hymenoptera: Apidae: Bombus spp.) from highlands in Bolivia and Peru

ABSTRACT Bumble bees of the genus Bombus spp. play a key role as effective pollinators in the biodiversity rich Central Andean region; however, parasites and pathogens may negatively influence their abundance and population numbers. In this study, we aimed at verifying the occurrence of these natural enemies in the Andes regions of Bolivia and Peru. Workers and males of Bombus funebris, Bombus opifex and Bombus pauloensis were collected in three localities from highlands at ~1,700 m.a.s.l. Analyses by both optical and molecular techniques revealed the presence of the Apis mellifera filamentous virus (AmFV), the microsporidium Vairimorpha ceranae, the flagellates Crithidia bombi and Lotmaria passim, and five species of mites in the genera Parasitellus Willmann, Pneumolaelaps Berlese, Proctolaelaps Berlese, Tyrophagus Oudemans, and Kuzinia Zachvatkin. Given the potential entry of the allochthonous bumble bee Bombus terrestris to the region, this information is essential to evaluate autochtonous biodiversity conservation strategies.

Thermally conductive molecular assembly composed of an oligo(ethylene glycol)-modified filamentous virus with improved solubility and resistance to organic solvents

Organic polymers are generally regarded as thermal insulators because of their random arrangement of polymeric chains that lead to scattering of heat-conducting phonons. We previously found that highly oriented assemblies composed of M13 phage, a filamentous virus, showed high thermal diffusivity, even though phonons are conducted on noncovalent bonds. However, biomolecular M13 phages did not allow utilization of organic solvents, resulting in limited applicability. Here, we utilized chemically modified M13 phage with oligo(ethylene glycol) (OEG) to improve its solubility and resistance to organic solvents, and to expand the applicability of phage-based thermally conductive assemblies. The high thermal diffusivity of assemblies composed of M13 phages modified with longer EG chains (EG unit: 10) was maintained when the assemblies were prepared using a mixed solvent of water and tetrahydrofuran, whereas that of assemblies composed of unmodified and shorter EG chain (3 and 6)-modified M13 phages was not maintained. When the mixed solvent was used, structural characterization revealed the presence of ordered and hexagonally packed structures that formed ordered assemblies, leading to phonon-conductive assemblies in the case of the longer EG chain-modified M13 phages. Our results will contribute to the construction of novel thermally conductive soft materials composed of biomacromolecular assemblies using organic solvents on substrates with complex surface morphologies and/or hydrophobic surfaces. Utilization of chemical modification of M13 phage with oligo(ethylene glycol) to improve its solubility and resistance to organic solvents successfully expands the applicability of M13 phage-based thermally conductive assemblies. The high thermal diffusivity of the assemblies composed of M13 phage modified with longer ethylene glycol chains was maintained when the assemblies were prepared using a mixed solvent of water and tetrahydrofuran. Our results will contribute to construction of novel thermally conductive soft materials composed of biomacromolecular assemblies using organic solvents on substrates with complex surface morphologies and/or hydrophobic surfaces.

Atomic structure of potato virus X, the prototype of the Alphaflexiviridae family.

Potato virus X (PVX) is a positive-sense single-stranded RNA (ssRNA) filamentous plant virus belonging to the Alphaflexiviridae family, considered in recent years as a tool for nanotechnology applications. We present the cryo-electron microscopy structure of the PVX particle at a resolution of 2.2 Å. The well-defined density of the coat proteins and of the genomic RNA allowed a detailed analysis of protein-RNA interactions, including those mediated by solvent molecules. The particle is formed by repeated segments made of 8.8 coat proteins, forming a left-handed helical structure. The RNA runs in an internal crevice along the virion, packaged in 5-nucleotide repeats in which the first four bases are stacked in the classical way, while the fifth is rotated and nearly perpendicular. The resolution of the structure described here suggests a mechanism for the virion assembly and potentially provides a platform for the rational design of antiviral compounds and for the use of PVX in nanotechnology.

Phoretic mites on South American bumblebees (Bombus spp.) as parasite carriers: a historical input

Phoretic mites associated with bumblebees are considered commensals that represent little or no risk for their hosts. The aim of the present study was to assess the potential role of the phoretic mites Pneumolaelaps longanalis and Parasitellus fucorum, as carriers of parasites that are known to affect bees. Mites were extracted from bumblebees dating between 1945 and 1981 which belong to museum collections, and from a freshly collected queen. The DNA was extracted and amplified, and the final products were sequenced and analyzed. Parasite’s DNA was found in all samples. Ascosphaera spp., Crithidia bombi, Lotmaria passim, and Nosema ceranae were present in both mite species. Moreover, DNA from Apis mellifera filamentous virus (AmFV), Apicystis bombi, Ascosphaera apis, Nosema apis, and Nosema bombi was found in P. longanalis. This study represents the oldest report of parasite’s DNA in bumblebee phoretic mites, highlighting the relevance of museum samples for historical studies in parasitology.

Complete genomic sequence of Noni mosaic virus (NoMV) associated with a mosaic disease in Morinda citrifolia L.

An outbreak of a virus-like disease has caused severe damage to noni plants (Morinda citrifolia L.) in Xishuangbanna area of Yunnan province in southwest China since 2015. The diseased plants displayed typical mosaic symptom with light and dark green patches on leaves. Flexuous filamentous virus particles of about 800 nm in length were observed from the leaf saps by transmission electron microscope. Illuemina transcriptomic sequencing further revealed the presence of a potyvirus and its near complete genome was obtained from de novo assembly. The complete genome of 9659 nts was obtained by Sanger sequencing of eight amplicons generate by RT-PCR and 5′ and 3’ RACE. BLASTp analysis of the polyprotein sequence showed that the virus was most closely related to Tobacco vein banding mosaic virus (TVBMV), but these two viruses only shared 50.7% amino acid sequence similarity. Both phylogenetic analyses of the polyprotein and CP amino acid sequences indicated that this virus is a member of genus Potyvirus. However, the low sequence homology with all known potyviruses established this virus as a new species in the genus, tentatively named as Noni mosaic virus (NoMV). Our field surveys showed that 100% of the symptomatic samples and 28.57% of the asymptomatic samples were infected with this novel potyvirus. Aphids collected from diseased leaves were also detected carrying the virus and aphid transmission test confirmed it can transmit the NoMV. In summary, our data indicated that a novel species of potyvirus, NoMV, is prevalent in Yunnan, China and is associated with an emerging mosaic disease on M. citrifolia.

A Novel Packing for A-Form DNA in an Icosahedral Virus

Studies on viruses infecting archaea living in the most extreme environments continue to show a remarkable diversity of structures, suggesting that the sampling continues to be very sparse. We have used electron cryo-microscopy (cryo-EM) to study at 3.7 Å resolution the structure of the Sulfolobus polyhedral virus 1 (SPV1) which was originally isolated from a hot, acidic spring in Beppu, Japan. The two capsid proteins with variant single jelly-roll folds form pentamers and hexamers which assemble into a T=43 icosahedral shell. In contrast to tailed icosahedral double-stranded DNA (dsDNA) viruses infecting bacteria and archaea, and herpesviruses infecting animals and humans, where naked DNA is packed under very high pressure due to the repulsion between adjacent layers of DNA, the circular dsDNA in SPV1 is fully covered with a viral protein forming a nucleoprotein filament with attractive interactions between layers. Most strikingly, we have been able to show that the DNA is in an A-form, as it is in the filamentous viruses infecting hyperthermophilic acidophiles. While previous studies have suggested that DNA is in the B-form in bacteriophages, we believe that this is the first direct visualization of the structure of DNA in an icosahedral virus.

Structure of a filamentous virus uncovers familial ties within the archaeal virosphere.

Viruses infecting hyperthermophilic archaea represent one of the most enigmatic parts of the global virome, with viruses from different families showing no genomic relatedness to each other or to viruses of bacteria and eukaryotes. Tristromaviruses, which build enveloped filamentous virions and infect hyperthermophilic neutrophiles of the order Thermoproteales, represent one such enigmatic virus families. They do not share genes with viruses from other families and have been believed to represent an evolutionarily independent virus lineage. A cryo-electron microscopic reconstruction of the tristromavirus Pyrobaculum filamentous virus 2 at 3.4 Å resolution shows that the virion is constructed from two paralogous major capsid proteins (MCP) which transform the linear dsDNA genome of the virus into A-form by tightly wrapping around it. Unexpectedly, the two MCP are homologous to the capsid proteins of other filamentous archaeal viruses, uncovering a deep evolutionary relationship within the archaeal virosphere.

Purification of Rice Stripe Virus.

Although many spherical and rod-shaped plant virus purification protocols are now available, only a few protocols on filamentous plant virus purification have been published. Here, we report a protocol for large-scale purification of Rice stripe virus (RSV) from RSV-infected rice tissues. RSV virions with high infectivity were first precipitated with polyethylene glycol (PEG) followed by pelleting through primary ultracentrifugation, ultracentrifugation in a glycerol cushion and ultracentrifugation in density gradient. The purified RSV virions can not only be viewed as filamentous particles under an electron microscope, but can also be acquired by insect vector through direct injection into insect body or through membrane feeding prior to transmission to rice plants.

Controlled assembly of filamentous viruses into hierarchical nano- to microstructures at liquid/liquid interfaces.

Recently, viruses have been regarded as useful molecular assemblies for materials applications rather than as disease-causing agents. The orderly assembled structures of the viruses are highly related to the resultant properties and functions of the assemblies; however, methods to control the assembly are still limited. Here, we demonstrated the assembly of filamentous viruses into hierarchical nano- to microstructures at liquid/liquid interfaces through emulsification in a controlled manner. The viruses form fibrous nanostructures of several micrometers length, which are much longer than the original virus. Subsequently, the fibers self-assemble into well-packed ordered microstructures. Furthermore, the resultant hierarchically assembled structures showed long-term stability and potential applicability through the desired functionalization.

First Report of Cassava common mosaic virus Infecting Cassava in Mainland China

First Report of <i>Cassava common mosaic virus</i> Infecting Cassava in Mainland China

First Report of Tobacco Vein Banding Mosaic Virus Naturally Infecting Black Nightshade (Solanum nigrum) in China

Black nightshade (Solanum nigrum L. var. humile [Bernh.]) is a common solanaceous plant that is an arable weed and also used in herbal medicine in China (Wang et al. 2017b; Zhao et al. 2018). The weed occurs commonly in a wide variety of habitats, including orchards, fields, villages, and roadsides in southwest provinces in China. In May 2019, about 3% of black nightshade plants growing in a tomato field at Kunming city in Yunnan province in China were found showing typical symptoms of viral disease (chlorosis, mosaic, leaf twisting, and plant stunting). To identify the suspected virus, one plant with severe symptoms was chosen for small RNA sequencing. Total RNA was extracted from leaves of symptomatic tissues by TruSeq Small RNA Sample Prep Kits (Illumina, San Diego, CA), and a small RNA library was constructed and then sequenced by high-throughput sequencing (HTS) on an Illumina HiSeq 2500 platform. The analysis generated approximately 11.37 million Illumina reads (15 to 40 nt in length), of which more than 0.082 million reads were mapped to the tobacco vein banding mosaic virus (TVBMV) genome (reference sequence accession no. NC_009994.1). No other viruses and viroids were detected. Further observation by transmission electron microscopy showed that the negatively stained crude sap of two symptomatic plants contained flexuous, filamentous virus particles about 600 to 900 nm in length, typical of a potyvirus. To further confirm the results, total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific, Carlsbad, CA), first-strand cDNA was synthesized from 1 µg of total RNA using an oligo dT primer and a random primer mix, and then fragments of the TVBMV-CP gene were amplified with specific primers (F, 5′-AATGACGAACAGACAGTTGATGCTG-3′; R, 5′-CACGCCACTCACACCAAGTAGG-3′; designed from the CP gene of an HTS-generated clone) by 2×T5 Super PCR Mix (Tsingke BioTech, China). After cloning into a pGEM-T vector (Promega Biotech, U.S.A.), six clones of each fragment were randomly chosen for Sanger sequencing (Tsingke BioTech). The clone sequences were identical, and this partial TVBMV sequence was then submitted to GenBank (accession no. MN331841). This new sequence was most similar to Chinese isolate XDBB-01 (95.1% nucleotide identity to the CP mRNA, accession MG880266.1, and 98.2% amino acid identity to the corresponding coat protein AYG97953.1). Ten additional black nightshade plants with similar symptoms and another three asymptomatic plants were randomly selected and tested for the presence of TVBMV using the same primers. All 10 symptomatic plants tested positive for TVBMV, whereas the asymptomatic plants tested negative by reverse transcription PCR (RT-PCR) (data not shown). Transmissibility of TVBMV from black nightshade to tomato (cultivar KP13) by aphids (Myzus persicae), which were present widely in the tomato fields in the region, was tested according to the method reported by Yu et al. (2007). At 21 days postinoculation, 10 out of 20 inoculated plants showed leaf twisting symptoms. RT-PCR confirmed the presence of TVBMV in these plants but not in plants without symptoms. Results from three biological repeats were consistent, demonstrating that black nightshade can serve as an effective inoculum source for TVBMV. TVBMV has been reported to infect tobacco, potato, Datura stramonium, wild eggplant, tomato, and sesame in China (Wang et al. 2017a). To our knowledge, this is the first report of TVBMV infecting S. nigrum in natural field conditions. Our findings indicate that S. nigrum in the field could be a reservoir of TVBMV to infect tomato and cause viral disease in tomato fields.

Occurrence and Molecular Phylogeny of Honey Bee Viruses in Vespids.

Since the discovery that honey bee viruses play a role in colony decline, researchers have made major breakthroughs in understanding viral pathology and infection processes in honey bees. Work on virus transmission patterns and virus vectors, such as the mite Varroa destructor, has prompted intense efforts to manage honey bee health. However, little is known about the occurrence of honey bee viruses in bee predators, such as vespids. In this study, we characterized the occurrence of 11 honey bee viruses in five vespid species and one wasp from four provinces in China and two vespid species from four locations in France. The results showed that all the species from China carried certain honey bee viruses, notably Apis mellifera filamentous virus (AmFV), Deformed wing virus (DWV), and Israeli acute paralysis virus (IAPV); furthermore, in some vespid colonies, more than three different viruses were identified. In France, DWV was the most common virus; Sacbrood virus (SBV) and Black queen cell virus (BQCV) were observed in one and two samples, respectively. Phylogenetic analyses of IAPV and BQCV sequences indicated that most of the IAPV sequences belonged to a single group, while the BQCV sequences belonged to several groups. Additionally, our study is the first to detect Lake Sinai virus (LSV) in a hornet from China. Our findings can guide further research into the origin and transmission of honey bee viruses in Vespidae, a taxon of ecological, and potentially epidemiological, relevance.

Microstructural Understanding of the Length- and Stiffness-Dependent Shear Thinning in Semidilute Colloidal Rods

Complex fluids containing low concentrations of slender colloidal rods can display a high viscosity, while little flow is needed to thin the fluid. This feature makes slender rods essential constituents in industrial applications and biology. Though this behaviour strongly depends on the rod-length, so far no direct relation could be identified. We employ a library of filamentous viruses to study the effect of rod size and flexibility on the zero-shear viscosity and shear-thinning behaviour. Rheology and small angle neutron scattering data are compared to a revised version of the standard theory for ideally stiff rods, which incorporates a complete shear-induced dilation of the confinement. While the earlier predicted length-independent pre-factor of the restricted rotational diffusion coefficient is confirmed by varying the length and concentration of the rods, the revised theory correctly predicts the shear thinning behaviour as well as the underlying orientational order. These results can be directly applied to understand the manifold systems based on rod-like colloids and design new materials.

Single-particle measurements of filamentous influenza virions reveal damage induced by freezing.

Clinical isolates of influenza virus produce pleiomorphic virions, ranging from small spheres to elongated filaments. The filaments are seemingly adaptive in natural infections, but their basic functional properties are poorly understood and functional studies of filaments often report contradictory results. This may be due to artefactual damage from routine laboratory handling, an issue which has been noted several times without being explored in detail. To determine whether standard laboratory techniques could damage filaments, we used immunofluorescence microscopy to rapidly and reproducibly quantify and characterize the dimensions of filaments. Most of the techniques we tested had minimal impact on filaments, but freezing to -70 °C, a standard storage step before carrying out functional studies on influenza viruses, severely reduced their concentration, median length and the infectivity of the whole virion population. We noted that damage from freezing is likely to have affected most of the functional studies of filaments performed to date, and to address this we show that it can be mitigated by snap-freezing or incorporating the cryoprotectant DMSO. We recommend that functional studies of filaments characterize virion populations prior to analysis to ensure reproducibility, and that they use unfrozen samples if possible and cryoprotectants if not. These basic measures will support the robust functional characterizations of filaments that are required to understand their roles in natural influenza virus infections.

Broad Geographic and Host Distribution of Apis mellifera Filamentous Virus in South American Native Bees

Abstract Apis mellifera filamentous virus (AmFV) is a large double stranded DNA virus of honey bees and its prevalence and relationship with other parasites is poorly known. Samples consisted of fifty-one adult bees belonging to eight native species collected using entomological nets in six provinces of Argentina, from 2009 to 2018. Total genomic DNA was extracted from individual bees and a 551 bp fragment of the Bro-N gene of AmFV was amplified by qPCR. In the present work we have reported for the first time both the presence and the wide geographic distribution of AmFV in Argentinian species of native bees. This is the first report of the presence of this virus associated with Xylocopa atamisquensis, X. augusti, X. frontalis, X. spendidula, Bombus pauloensis and Peponapis fervens. Detecting pathogens that could threaten native bee health is of outmost importance to generate both conservation and management strategies.

First molecular detection of Apis mellifera filamentous virus (AmFV) in honey bees (Apis mellifera) in Argentina

Although many honey bee RNA viruses have been correlated with colony declines, little is known regarding the potential role of DNA viruses. Apis mellifera filamentous virus (AmFV) is a large double stranded DNA virus of honey bees and its prevalence and relationship with other parasites is poorly known. In the present work, we have described a high prevalence of AmFV in Argentinian apiaries, and a retrospective survey of archived honey bee samples revealed that AmFV has been present in Argentina since at least 2006. This is the first report involving AmFV in South America.