Purification and Identification of a Rhabdovirus from Thunbergia alata

A subgroup 2 rhabdovirus was isolated in south-east Queensland from black-eyed Susan (Thunbergia alata) with symptoms of vein yellowing, vein clearing and leaf distortion. Bacilliform particles accumulated in the perinuclear space of infected plants and measured 69 ± 7 × 161 ± 8 nm in unfixed preparations. The virus was not transmitted mechanically. Purified preparations of the Thunbergia alata rhabdovirus (TaRV) contained four major proteins with molecular weights of 80 kD, 48 kD, 40 kD and 35 kD, similar to those of datura yellow vein virus (DYW), a newly described rhabdovirus from Australia. The 80 kD protein was identified as the viral glycoprotein. In immunoblots, the two largest proteins of TaRV reacted strongly with antiserum to DYW, but were serologically distinct from sonchus yellow net, cereal chlorotic mottle, potato yellow dwarf and lettuce necrotic yellows viruses. TaRV is considered to be a strain of DYW.     

The occurrence of maize mosaic virus on sorghum in India1

A leaf disease of sorghum (Sorghum bicolor) characterised by fine discontinuous chlorotic streaks between the veins, was observed on sorghum grown during the 1987/88 post-rainy season in peninsular India. Early-infected plants were stunted, had shortened internodes, and produced poorly developed panicles. The virus was transmitted by the delphacid planthopper, Peregrinus maidis. Negatively stained leaf dip preparations contained bullet-shaped virus particles (208 ± 4.4 × 66 ± 1.0 nm) resembling those of rhabdoviruses. In ultrathin sections, the particles budded through the inner nuclear membrane and were present in the cytoplasm within membrane-bound vesicles that were apparently contiguous with the distended outer nuclear membrane. A method for purifying the virus was developed utilising polyethylene glycol (PEG) precipitation, Celite filtration and sucrose densitygradient centrifugation. An antiserum was produced in rabbits with a titre of 1/2650 in the precipitin ring interphase test. The virus could be detected in infected sorghum leaf tissues using a direct antigen coating form of enzyme-linked immunosorbent assay (DAC-ELISA). In immuno-double diffusion tests, the virus reacted positively with antisera to maize mosaic virus (MMV) from Reunion (MMV-RN) and Hawaii (MMV-HI), but not with antisera to barley yellow striate mosaic (BYSMV), cereal chlorotic mottle (CCMV), and cynodon chlorotic streak (CCSV) viruses. Thus, the virus isolated from sorghum is designated the MMV-S isolate. In DAC-ELISA tests, MMV-S reacted positively with antisera to MMV-R, MMV-HI, MMV-Florida isolate, CCSV, and CCMV, and weakly with antiserum to BYSMV. SDS-polyacrylamide gel electrophoresis revealed four major proteins of relative mass M_r 70 000, 59 000, 32 000 and 28 000. In electro-blot immunoassay, MMV and CCSV antisera detected the G and N proteins. These data suggest that MMV-S should be placed in the sonchus yellow net virus subgroup of plant rhabdoviruses.     

Plant-produced recombinant influenza vaccine based on virus-like HBc particles carrying an extracellular domain of M2 protein

Conventional influenza vaccines are based on a virus obtained in chicken embryos or its components. The high variability of the surface proteins of influenza virus, hemagglutinin and neuraminidase, requires strain-specific vaccines matching the antigenic specificity of newly emerging virus strains to be developed. A recombinant vaccine based on a highly conservative influenza virus protein M2 fused to a nanosized carrier particle can be an attractive alternative to traditional vaccines. We have constructed a recombinant viral vector based on potato X virus that provides for expression in the Nicotiana benthamiana plants of a hybrid protein M2eHBc consisting of an extracellular domain of influenza virus M2 protein (M2e) fused to hepatitis B core antigen (HBc). This vector was introduced into plant cells by infiltrating leaves with agrobacteria carrying the viral vector. The hybrid protein M2eHBc was synthesized in the infected N. benthamiana plants in an amount reaching 1–2% of the total soluble protein and formed virus-like particles with the M2e peptide presented on the surface. Methods of isolation and purification of M2eHBc particles from plant producers were elaborated. Experiments on mice have shown a high immunogenicity of the plant-produced M2eHBc particles and their protective effect against lethal influenza challenge. The developed transient expression system can be used for production of M2e-based candidate influenza vaccine in plants.     

Interactions between the tomato spotted wilt virus movement protein and plant proteins showing homologies to myosin, kinesin and DnaJ-like chaperones

The non-structural protein encoded by the M RNA segment (NSm) of tomato spotted wilt virus (TSWV) has been implicated in cell-to-cell movement of nucleocapsids through modified plasmodesmata. Recently, DnaJ-like proteins from Nicotiana tabacum (tobacco) and Arabidopsis thaliana have been identified as NSm interacting host proteins, implying an involvement of molecular chaperones during systemic spread of the virus or other, presently unknown NSm-mediated virus functions. Examination of additional TSWV host plants and improvement of yeast two-hybrid interaction trap experiments led to the isolation of a DnaJ-like protein from Lycopersicon esculentum (tomato) and the identification of a protein from A. thaliana sharing some homologies with myosin and kinesin-like polypeptides. Sequence alignments of the tomato DnaJ-like protein unveiled the corresponding gene as an orthologue to the tobacco and A. thaliana DnaJ genes, substantiating that NSm interacting DnaJ-like polypeptides, identified from three different TSWV host species, apparently form a subgroup distinct from archetypical DnaJ chaperones. Increased levels of DnaJ-like proteins could be detected in TSWV systemically infected leaves and in plants exposed to heat shock, showing that the NSm interacting DnaJ-like chaperones are inducible upon biotic and abiotic stress. All together, the identification of DnaJ-like proteins and a protein resembling myosin and kinesin as NSm interacting plant proteins is in accordance with results accomplished for movement proteins from other plant attacking viruses showing an involvement of molecular chaperones and the cytoskeleton in at least intracellular trafficking.     

Eggplant Mottled Dwarf Virus Associated with Vein Yellowing of Honeysuckle

A rhabdovirus isolated in Tunisia by mechanical inoculation from honeysuckle (Lonicera sp.) plants with vein yellowing, was compared with a Tunisian isolate of eggplant mottled dwarf virus (EMDV). The two viruses had similar host ranges and caused the same symptoms in artificially infected hosts. The honeysuckle virus induced in eggplant a syndrome indistinguishable from that typical of EMDV. The two viruses could not be differentiated serologically, had particles of the same size and elicited identical cytopathological alterations in naturally and artificially infected hosts. Honeysuckle is the first host, besides eggplant, found to be naturally infected with EMDV.     

Identification of a movement protein of rice yellow stunt rhabdovirus

Rice yellow stunt rhabdovirus (RYSV) encodes seven genes in its negative-sense RNA genome in the order 3'-N-P-3-M-G-6-L-5'. The existence of gene 3 in the RYSV genome and an analogous gene(s) of other plant rhabdoviruses positioned between the P and M genes constitutes a unique feature for plant rhabdoviruses that is distinct from animal-infecting rhabdoviruses in which the P and M genes are directly linked. However, little is known about the function of these extra plant rhabdovirus genes. Here we provide evidence showing that the protein product encoded by gene 3 of RYSV, P3, possesses several properties related to a viral cell-to-cell movement protein (MP). Analyses of the primary and secondary protein structures suggested that RYSV P3 is a member of the "30K" superfamily of viral MPs. Biolistic bombardment transcomplementation experiments demonstrated that RYSV P3 can support the intercellular movement of a movement-deficient potexvirus mutant in Nicotiana benthamiana leaves. In addition, Northwestern blot analysis indicated that the RYSV P3 protein can bind single-stranded RNA in vitro, a common feature of viral MPs. Finally, glutathione S- transferase pull-down assays revealed a specific interaction between the RYSV P3 protein and the N protein which is a main component of the ribonucleocapsid, a subviral structure believed to be involved in the intercellular movement of plant rhabdoviruses. Together, these data suggest that RYSV P3 is likely a MP of RYSV, thus representing the first example of characterized MPs for plant rhabdoviruses.     

A proline-rich motif within the matrix protein of vesicular stomatitis virus and rabies virus interacts with WW domains of cellular proteins: implications for viral budding

The matrix (M) protein of rhabdoviruses has been shown to play a key role in virus assembly and budding; however, the precise mechanism by which M mediates these processes remains unclear. We have associated a highly conserved, proline-rich motif (PPxY or PY motif, where P denotes proline, Y represents tyrosine, and x denotes any amino acid) of rhabdoviral M proteins with a possible role in budding mediated by the M protein. Point mutations that disrupt the PY motif of the M protein of vesicular stomatitis virus (VSV) have no obvious effect on membrane localization of M but instead lead to a decrease in the amount of M protein released from cells in a functional budding assay. Interestingly, the PPxY sequence within rhabdoviral M proteins is identical to that of the ligand which interacts with WW domains of cellular proteins. Indeed, results from two in vitro binding assays demonstrate that amino acids 17 through 33 and 29 through 44, which contain the PY motifs of VSV and rabies virus M proteins, respectively, mediate interactions with WW domains of specific cellular proteins. Point mutations that disrupt the consensus PY motif of VSV or rabies virus M protein result in a significant decrease in their ability to interact with the WW domains. These properties of the PY motif of rhabdovirus M proteins are strikingly analogous to those of the late (L) budding domain identified in the gag-specific protein p2b of Rous sarcoma virus. Thus, it is possible that rhabdoviruses may usurp host proteins to facilitate the budding process and that late stages in the budding process of rhabdoviruses and retroviruses may have features in common.     

Auswirkungen des Anbaus einer Wirts- und einer Nichtwirtspflanze unter wechselnden ökologischen Bedingungen auf die Populationsdynamik von Rhizoctonia solani Kühn und Fusarium solani f. pisi (Jones) Snyd. et Hans

An isometric virus was isolated from Helianthus annuus L. plants showing a yellow leaf spot mosaic on affected leaves. Infected plants were found in different ecological regions of Ukraine. A procedure of virus purification is described. The diametres of the virus particles were nonuniform and ranged from 50 to 120 nm. The sedimentation coefficient of the virus was 518–540 S and the floating density in the CsCl gradient was 1.22 g/cm³. The MW of proteins separated by electrophoresis amounted to 78±0.9, 58±0.8, 52±0.2, and 27±0.8 kDa, respectively. The virus was assigned to the tospoviruses for which sunflower is a new previously undescribed natural host plant.     

Characterisation of datura yellow vein virus, a newly described rhabdovirus from Australia

A previously undescribed sub-group 2 rhabdovirus was isolated in Queensland from Datura stramonium with symptoms of vein yellowing, leaf distortion and reduced leaf size. Particles accumulated in the perinuclear space of infected cells of D. stramonium and measured 77 × 166 nm in preparations from sap. The virus was named datura yellow vein virus (DYVV) and was graft-transmitted to several hosts in the Solanaceae including Lycopersicon esculentum, Nicotiana tabacum and Solanum melongena, but not to Capsicum annuum or Solanum tuberosum. DYVV was not transmitted by mechanical inoculation and no insect vector was found. Purified particles of DYVV contained four structural proteins with molecular weights of about 78, 47, 41 and 36 kd. The 78 kd protein bound the lectin concanavalin A, thus identifying it as the viral glycoprotein. DYVV was serologically distinct from 11 other rhabdoviruses belonging to both subgroups, including potato chlorotic stunt, potato yellow dwarf (2 isolates) and tomato vein yellowing viruses. The glycoprotein only of DYVV cross-reacted with a polyclonal antiserum to sonchus yellow net virus.     

Sigma viruses from three species of Drosophila form a major new clade in the rhabdovirus phylogeny

The sigma virus (DMelSV), which is a natural pathogen of Drosophila melanogaster, is the only Drosophila-specific rhabdovirus that has been described. We have discovered two new rhabdoviruses, D. obscura and D. affinis, which we have named DObsSV and DAffSV, respectively. We sequenced the complete genomes of DObsSV and DMelSV, and the L gene from DAffSV. Combining these data with sequences from a wide range of other rhabdoviruses, we found that the three sigma viruses form a distinct clade which is a sister group to the Dimarhabdovirus supergroup, and the high levels of divergence between these viruses suggest that they deserve to be recognized as a new genus. Furthermore, our analysis produced the most robustly supported phylogeny of the Rhabdoviridae to date, allowing us to reconstruct the major transitions that have occurred during the evolution of the family. Our data suggest that the bias towards research into plants and vertebrates means that much of the diversity of rhabdoviruses has been missed, and rhabdoviruses may be common pathogens of insects.     

Characterization of Maize Iranian Mosaic Virus and Comparison with Hawaiian and Other Isolates of Maize Mosaic Virus (Rhabdoviridae)

Maize Iranian mosaic virus (MIMV) was characterized and compared with isolates of Maize mosaic virus (MMV, genus Nucleorhabdovirus, family Rhabdoviridae) in insect transmission, cytopathology and ultrastructure of infected maize cells, virion proteins and serologically. MIMV is naturally transmitted by Ribautodelphax notabilis, a delphacid planthopper, in Iran. In this study, another planthopper, Peregrinus maidis, vector of MMV, transmitted MIMV with an estimated efficiency of 0.4–1.6% following feeding on MIMV‐infected maize plants and 64% following injection of MIMV into the hemolymph, suggesting that P. maidis gut tissues largely blocked MIMV transmission. MIMV and MMV‐HI (Hawaii) induced similar cytopathologies in cells of infected maize leaves, with virions budding through inner nuclear and endoplasmic reticulum membranes. In thin sections, virions of MIMV were significantly shorter than those of MMV‐HI. Sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis of virions of MIMV, MMV‐HI, MMV‐CR (Costa Rica) and MMV‐FL (Florida) yielded six proteins of which four were identified as the putative G, N, P and M proteins of plant rhabdoviruses. The N, P and M proteins of MIMV migrated faster in gels than those of the MMV isolates indicating a lower molecular weight, whereas the bands corresponding to the G proteins migrated similarly for both viruses. Polyclonal antibodies to MMV‐HI failed to react with virions of MIMV in enzyme‐linked immunosorbent assay (ELISA) and with MIMV proteins in Western blots. In contrast, these antibodies reacted strongly with MMV‐HI and MMV‐FL virions in ELISA and with MMV‐HI, MMV‐CR and MMV‐FL proteins in Western blots. Further, in ELISA, polyclonal antibodies to MMV‐MR (Mauritius) reacted weakly with MIMV virions but strongly with MMV‐HI and MMV‐FL virions. Thus, it is concluded that MIMV is a new virus of the Nucleorhabdovirus genus that may be distantly related to MMV.     

Pathology and properties of tulip virus X, a new potexvirus

Tulip virus X (TVX), a previously undescribed mechanically transmissible virus, causes chlorotic and necrotic lesions in leaves and streaks of intensified pigmentation in tepals of tulip plants. The virus infected 22 of 42 other plant species in 10 of 14 families, but most host species were infected only erratically. TVX is best propagated in Chenopodium quinoa and assayed in C. amaranticolor. Spindleshaped inclusions were observed in epidermal cells of C. amaranticolor leaves. Leaf extracts from C. quinoa contained flexuous filamentous particles measuring c. 495 ×13 nm. The extracts were infective after dilution to 10^-9, after heating for 10 min at 60 °C but not at 65 °C, and after storage at c. 20 °C for 30 days or at -20 °C for 6 months. TVX particles were purified (500 μg/g C. quinoa leaf) from tissue extracts in 0.067 M phosphate buffer containing 10 mM EDTA at pH 7, by twice precipitating the virus with 8% polyethylene glycol in 0.2 M NaCl followed by differential centrifugation. The virus particles have a sedimentation coefficient (s_{20, w}) of 102 S. They contain a protein of mol. wt c. 22 500 and a nucleic acid that, when glyoxalated, migrates in agarose gel like single-stranded RNA of mol. wt 2.05 × 10⁶. TVX particles tend to aggregate, and evidence was obtained that a 118 S component which was consistently observed in purified preparations and in infective sap is an end-to-end dimer. A distant serological relationship was found between particles of TVX and those of viola mottle and hydrangea ringspot viruses, but no serological relationship was detected to nine other potexviruses. TVX is considered to be a distinct and definitive member of the potexvirus group.     

Infection of potato plants with potato leafroll virus changes attraction and feeding behaviour of Myzus persicae

Potato leafroll virus (PLRV; genus Polerovirus, family Luteoviridae) is a persistently transmitted circulative virus that depends on aphids for spreading. The primary vector of PLRV is the aphid Myzus persicae (Sulzer) (Homoptera: Aphididae). Solanum tuberosum L. potato cv. Kardal (Solanaceae) has a certain degree of resistance to M. persicae: young leaves seem to be resistant, whereas senescent leaves are susceptible. In this study, we investigated whether PLRV‐infection of potato plants affected aphid behaviour. We found that M. persicae's ability to differentiate headspace volatiles emitted from PLRV‐infected and non‐infected potato plants depends on the age of the leaf. In young apical leaves, no difference in aphid attraction was found between PLRV‐infected and non‐infected leaves. In fact, hardly any aphids were attracted. On the contrary, in mature leaves, headspace volatiles from virus infected leaves attracted the aphids. We also studied the effect of PLRV‐infection on probing and feeding behaviour (plant penetration) of M. persicae using the electrical penetration graph technique (DC system). Several differences were observed between plant penetration in PLRV‐infected and non‐infected plants, but only after infected plants showed visual symptoms of PLRV infection. The effects of PLRV‐infection in plants on the behaviour of M. persicae, the vector of the virus, and the implications of these effects on the transmission of the virus are thoroughly discussed.     

Purification and properties of lucerne Australian symptomless virus, a new virus infecting lucerne in Australia

A virus with isometric particles c. 26–28 nm in diameter isolated from naturally infected lucerne (Medicago sativa) in Australia and reported there to be a strain of lucerne Australian latent virus (LALV), is shown to be a distinct virus. The virus, called lucerne Australian symptomless (LASV), was mechanically transmitted to 10 of 22 plant species inoculated, but only induced symptoms in three Chenopodium species and Gomphrena globosa. Virus particles occurred in relatively low concentrations in plant sap, and the virus could not be reliably maintained in culture by serial transmission to plants during winter (October-April). During the summer, sap of infected C. quinoa remained infective after diluting 10^-2 but not 10^-3, after heating for 10 min at 50 but not 55 ^oC and after storage for 24 days (the longest period tested) at 20, 4 and -15 ^oC. LASV was seed-borne to 6% of C. quinoa seedlings. Partially purified preparations of virus particles contained one nucleoprotein component with a sedimentation coefficient of c. BOS. Particles contained two polypeptide species of estimated mol. wts 26 000 and 40 000, and two ssRNA species which, when denatured in glyoxal, had apparent mol. wts of 2–5 times 10⁶ and 1–4 times 10⁶. The infectivity of virus RNA was abolished by incubation with proteinase K. Purified particles of LASV reacted with homologous antiserum (gel diffusion titre 1:256) but not with antiserum to LALV or to 13 other plant viruses with isometric particles including arracacha B (AVB), broad bean wilt, rubus Chinese seed-borne (RCSV) and strawberry latent ringspot (SLRV) viruses, and five comoviruses. These properties distinguish LASV from LALV and from all recognised nepoviruses and comoviruses. Its closest affinities are with SLRV, RCSV and possibly AVB; these viruses may comprise a distinct virus group or nepovirus subgroup.     

Transmission,Particle Size and Additional Hosts of the Rhabdovirus Causing Maize Mosaic in Shiraz,Iran1

The rhabdovirus causing maize mosaic in Shiraz, Iran, is transmitted by Ribautodelphax notabilis Logvinenko (Homoptera, Delphacidae). Average size of bullet-shaped virus particles in negatively stained leaf-dip preparations of naturally or experimentally infected plants was 81 × 179 nm. The virus is transmitted to wheat and barley causing mosaic and severe stunting. Similar virus particles have been observed in leaf-dip preparations of naturally infected wheat, barley and Sudangrass. This is believed to be the first record of the involvement of R. notabilis in virus transmission. The relationship of the described isolate with similar viruses infecting gramineous plants is discussed.     

The Effect of Arachidonic Acid and Viral Infection on the Phytohemagglutinin Activity during the Development of Tobacco Acquired Resistance

The effects of arachidonic acid (AA) on the development of viral infection and the activity of phytohemagglutinins in Nicotiana tabacum L. plants were studied. Cv. Samsun NN was used, which displayed a genotypically determined hypersensitive response to tobacco mosaic virus (TMV) infection. When tobacco leaf disks were treated with 10^–9 to –10^–7 M AA, viral reproduction was suppressed by 90–100%. The AA concentration of 10^–8 M was optimal for the improvement of plant virus resistance. Tobacco leaves maintained virus resistance for at least two weeks. Both AA treatment and TMV inoculation were accompanied by an enhanced lectin activity, which may indicate the involvement of lectins in the development of plant defense responses. Lectin accumulation was observed in the intact plants developing systemic resistance and in the detached leaves characterized by local resistance.     

Isolation of a lethal rhabdovirus from the cultured Chinese sucker Myxocyprinus asiaticus

A rhabdovirus was found to be associated with a lethal hemorrhagic disease in the cultured Chinese sucker Myxocyprinus asiaticus Bleeker. The rhabdovirus was amplified and isolated from the infected GCO (grass carp ovary) cells. In ultrathin sections of liver cells from the diseased fish, the virus particles exhibited the characteristic bacilliform morphology, and budded through vesicle membranes of the infected cells. The isolated rhabdovirus particles were found to have a bacilliform morphology with 2 rounded ends rather than a typical flat base. The virus particles were measured and ranged in size from 150 to 200 nm in length and 50 to 60 nm in diameter. Most other characteristics, including their size, extensive virus infectivity to fish cell lines, strong cytopathogenic effects, stability at high temperatures, vesicle formation in infected cells, structure protein electrophoretic patterns and the presence of an RNA genome, very closely resembled those of other fish rhabdoviruses. At present it is not known if this is a novel virus species or if it is an isolate of a known fish rhabdovirus. Until a confirmed identification can be made, we will temporarily refer to this virus as Chinese sucker rhabdovirus (CSRV).     

Purification and properties of cassava green mottle, a previously undescribed virus from the Solomon Islands

A sap-transmissible virus obtained from cassava with a green mottle disease occurring at Choiseul, Solomon Islands, was transmitted to 30 species in 12 plant families and was readily seed-borne in Nicotiana clevelandii. In cassava plants infected by inoculation with sap, the first leaves to be infected systemically developed a mottle with some necrosis whereas leaves produced subsequently were symptomless but contained the virus. Most other species developed chlorotic or necrotic local lesions and systemic mottle or necrosis. This was followed, in several species, by production of small symptomless virus-containing leaves. The virus was cultured in N. clevelandii; Chenopodium quinoa was used for local-lesion assays. Leaf extracts from infected N. clevelandii were infective after dilution to 10–⁵ but usually not at 10–⁶, after heating for 10 min at 60°C but not at 65°C, and after storage at 20°C for at least 12 days. The virus has isometric particles of 26 nm diameter which sediment as three components, all containing a protein of mol. wt c. 53000. The two fastest sedimenting components respectively contain single-stranded RNA of mol. wt, estimated after glyoxylation, c. 2.9 × 10⁶ and 2.3 × 10⁶. Both RNA species are needed for infection of plants. In tests with antiserum prepared to purified virus particles, the virus was detected in cassava and N. clevelandii by gel-diffusion precipitin tests, by immunosorbent electron microscopy and by ELISA. Despite its similarity to nepoviruses, the virus did not react with antisera to 18 members of the group. It was named cassava green mottle virus and is considered to be a previously undescribed nepovirus.     

Properties of broad bean yellow band virus, a possible new tobravirus

A virus was transmitted from broad bean plants in Apulia (Southern Italy) with leaves showing yellow rings, line patterns or yellow vein banding and malformations and necrosis of pods. Symptoms in some, but not all, test plants were similar to those induced by tobraviruses. Purified virus preparations contained two classes of rod-shaped particles containing c. 5% nucleic acid with sedimentation coefficients of 186S and 276S. After centrifugation to equilibrium in CsCl gradients, two components were resolved, with buoyant densities of 1·298 and 1·316 g/cm³. Unfractionated virus preparations contained two species of single-stranded RNA with mol. wts of c. 1·06 × 10⁶ and 2·48 × 10⁶ and one species of coat protein with mol. wt of c. 21 300. The modal lengths of the two classes of particles, both in plant sap and in purified preparations, were 77 nm (S particles) and 202 nm (L particles). L particles accumulated in infected cells in paracrystalline aggregates, whereas S particles were randomly distributed in the cytoplasm of cells. The virus was serologically unrelated to two isolates of tobacco rattle virus and two isolates of pea early-browning virus. The virus, named broad bean yellow band, is considered a distinct tobravirus.