Apple mosaic virus isolated from hop plants in Japan

A strain of apple mosaic virus was isolated from hop plants in Japan. The virus was purified from young hop plants and back-inoculated to virus-free hop plants obtained by meristem tip culture. Inoculated plants developed chlorotic spots, ringspots and a band pattern accompanied by necrosis in the inoculated and systemically infected leaves. Shoot tips of infected plants sometimes became necrotic and these symptoms resembled those of a ring- and band-pattern mosaiclike disease prevalent in hop gardens in Japan. Since apple mosaic virus was recovered from infected plants, it is likely that the virus was the causal agent of this disease. Agar gel double diffusion tests and ELISA showed the hop virus to be serologically closely related to apple mosaic virus (ApMV), and distantly related to prunus necrotic ringspot virus (PNRSV). The virus had a narrow host range, and infected only cucumber of 18 species of Cucurbitaceae, Chenopodiaceae, Leguminosae or Solanaceae inoculated. It produced chlorotic spots on the inoculated cotyledons of cucumber, but no systemic infection. By contrast, ApMV from apple and PNRSV from peach had wide host ranges and infected cucumber plants systemically.     

Antioxidative responses in Vitis vinifera infected by grapevine fanleaf virus

The antioxidative response of grapevine leaves (Vitis vinifera cv. Trebbiano) affected by the presence of grapevine fanleaf virus was studied during the summer of 2010 at three different harvest times (July 1st and 26th, and August 30th). At the first and second harvest, infected leaves showed increases in the concentration of superoxide radical and hydrogen peroxide, the latter increasing for enhanced activity of superoxide dismutase. In contrast, at the last harvest time, increases in the ascorbate pool and ascorbate peroxidase activity maintained hydrogen peroxide to control levels. The glutathione pool was negatively affected as summer progressed, showing a decrease in its total and reduced form amounts. At the same time, increases in the ascorbate pool were observed, making antioxidant defenses of grapevine effective also at the last harvest time. Increases in phenolic acids, and in particular in p-hydroxybenzoic acid, at the first and second harvest might have enhanced the efficiency of the antioxidant system through an interrelation between a peroxidase/phenol/ascorbate system and the NADPH/glutathione/ascorbate cycle. The lack of increase in p-hydroxybenzoic acid at the third harvest could be due instead to the enhanced utilization of this acid for hydrogen peroxide detoxification. With time, grapevine plants lost their capacity to contrast the spread of grapevine fanleaf virus, but acquired a greater ability to counteract pathogen-induced oxidative stress, being endowed with more reduced antioxidant pools.     

Transmission of the hop strain of arabis mosaic virus by Xiphinema diversicaudatum*

Hop plants became infected with the hop strain of arabis mosaic virus (AMV(H)) when grown in hopfield and woodland soil in which infected plants had been growing. Infection occurred in soil infested with the dagger nematode Xiphinema diversicaudatum, but neither in uninfested soil nor in soil previously heated to kill nematodes. X. diversicaudatum transferred direct from hop soils transmitted AMV(H) to young herbaceous plants and to hop seedlings; some of the hop seedlings developed nettlehead disease. A larger proportion of plants was infected using X. diversicaudatum obtained from a woodland soil and then given access to the roots of hop or herbaceous plants infected with AMV(H). AMV(H) was transmitted by adults and by larvae, in which the virus persisted for at least 36 and 29 wk, respectively. Difficulties were encountered in detecting AMV(H) in infected hop plants, due partly to the delay in virus movement from roots to shoots. Infection of hop shoots was seldom detected until the year after the roots were infested and sometimes nettlehead symptoms did not appear until the third year. Isolates of arabis mosiac virus from strawberry did not infect hop. The results are discussed in relation to the etiology and control of nettlehead and related diseases of hop.     

Strains of Prunus necrotic ringspot virus in hop (Humulus lupulus L.)

Purified preparations of Prunus necrotic ringspot virus (NRSV) from hop plants formed two light-scattering zones when centrifuged in sucrose density gradients; the upper and lower zones contained particles 25 mμ and 31 mμ in diameter respectively whose sedimentation coefficients were 79 S and 107 S. NSRV isolates from hop were of two distinct serological types: ‘A’ strains, serologically very closely related to NRSV isolates from apple; and ‘C’ strains more nearly related to NRSV from cherry. The variety Fuggle is tolerant to hop mosaic (not related to NRSV) and different selections of apparently healthy female plants usually contained A strains; but C strains were usually isolated from nettlehead-diseased plants. Either A or C strains occurred in male plants grown with the hop-mosaic tolerant varieties. In mosaic-sensitive varieties (Goldings and Bramlings) apparently healthy female plants tested were usually infected with C strains; either A or C types occurred in mosaic-sensitive male plants. NRSV was not detected in the seventy-four hop seedlings obtained from virus-infected plants. Some varieties developed nettlehead when infected with NRSV (A) or (C) + the hop form of arabis mosaic virus, but not with NRSV (A) or (C) alone. Others developed nettlehead when infected with arabis mosaic virus + NRSV (C) but not with arabis mosaic + NRSV (A). A and C strains can multiply together in the same hop plant. There is evidence of partial antagonism, however, and the fluctuating behaviour of the nettlehead syndrome probably reflects changes in the relative concentration of the two serotypes.     

Molecular biological properties of new isolates of plum pox virus strain Winona

Plum pox virus (PPV, genus Potyvirus, family Potyviridae) is the most important virus pathogen of stone fruit cultures of the genus Prunus from an economical standpoint. Winona strain (PPV-W) is the most variable of nine known virus strains and one of the most widespread in the European part of Russia. Six new PPV-W isolates were first discovered in green plantations of Moscow (Kp2U, Avang, Pulk, Pulk-1), in Taldomsky district of Moscow region (Karm), and in Kovrovsky district of Vladimir region (Vlad-4) on wild trees of plum Prunus domestica. 3’-Terminal segment of genome of new isolates was notable for high variability level. The study on the relationship with other isolates of this strain by means of phylogenetic analysis of gene sequence of the coat protein showed the lack of clusterization of Russian PPV-W isolates according to geographical principle. Inoculation of Nicotiana benthamiana plants by hop plant louse Phorodon humili from plum trees infected with Avang and Pulk isolates and by thistle aphid Brachycaudus cardui from the tree infected with Kp2U isolate led to the systemic viral infection in indicator plants, suggesting the possibility of PPV-W spread by both aphid species in nature. Transmission of PPV-W through seeds was not observed.     

Grapevine fanleaf virus (GFLV)-specific antibodies confer GFLV and Arabis mosaic virus (ArMV) resistance in Nicotiana benthamiana

Grapevine fanleaf virus (GFLV) is one of the most destructive pathogens of grapevine. In this study, we generated monoclonal antibodies binding specifically to the coat protein of GFLV. Antibody FL₃, which bound most strongly to GFLV and showed cross-reactivity to Arabis mosaic virus (ArMV), was used to construct the single-chain antibody fragment scFvGFLVcp-55. To evaluate the potential of this single-chain variable fragment (scFv) to confer antibody-mediated virus resistance, transgenic Nicotiana benthamiana plants were generated in which the scFv accumulated in the cytosol. Recombinant protein levels of up to 0.1% total soluble protein were achieved. The T₁ and T₂ progenies conferred partial or complete protection against GFLV on challenge with the viral pathogen. The resistance to GFLV in transgenic plants was strictly related to scFvGFLVcp-55 accumulation levels, confirming that the antibody fragment was functional in planta and responsible for the GFLV resistance. In addition, transgenic plants conferring complete protection to GFLV showed substantially enhanced tolerance to ArMV. We demonstrate the first step towards the control of grapevine fanleaf degeneration, as scFvGFLVcp-55 could be an ideal candidate for mediating nepovirus resistance.     

Vector Capability of Xiphinema americanum sensu lato in California

Seven field populations of Xiphinema americanum sensu lato from California''s major agronomic areas were tested for their ability to transmit two nepoviruses, including the prune brownline, peach yellow bud, and grapevine yellow vein strains of tomato ringspot virus and the bud blight strain of tobacco ringspot virus. Two field populations transmitted all isolates, one population transmitted all tomato ringspot virus isolates but failed to transmit bud blight strain of tobacco ringspot virus, and the remaining four populations failed to transmit any virus. Only one population, which transmitted all isolates, bad been associated with field spread of a nepovirus. As two California populations of Xiphinema americanum sensu lato were shown to have the ability to vector two different nepoviruses, a nematode taxonomy based on a parsimony of virus-vector relationship is not practical for these populations. Because two California populations of X. americanum were able to vector tobacco ringspot virus, commonly vectored by X. americanum in the eastern United States, these western populations cannot be differentiated from eastern populations by vector capability tests using tobacco ringspot virus.     

A strain of Holmes' ribgrass virus occurring in Yugoslavia

A virus having 300 nm long rod-shaped particles was isolated fromPlantago media L. in Yugoslavia. The virus was transmitted to 15 species of host plants the symptoms of which are described in detail. The symptoms corresponded to those that appeared after infection by the original Holmes' ribgrass virus (HRV). The investigated virus was compared both with the common strain of tobacco mosaic and the original Holmes' ribgrass viruses by means of serological tests. The agar double-diffusion tests showed that it is closely related to HRV and remotely related to the common strain of TMV. On the basis of these results we concluded that this virus represented a strain of the HRV. The investigations of the cell inclusions showed that our virus produced rounded plates instead of hexagonal prisms. Electron micrographs of ultrathin sectioned material demonstrated that these plates were formed by virus particles lying perpendicularly to the layers of the plates. The presence of plates also points to the fact that the investigated virus belongs to the HRV.     

Localization and subcellular association of Grapevine Pinot Gris Virus in grapevine leaf tissues

Despite the increasing impact of Grapevine Pinot gris disease (GPG-disease) worldwide, etiology about this disorder is still uncertain. The presence of the putative causal agent, the Grapevine Pinot Gris Virus (GPGV), has been reported in symptomatic grapevines (presenting stunting, chlorotic mottling, and leaf deformation) as well as in symptom-free plants. Moreover, information on virus localization in grapevine tissues and virus-plant interactions at the cytological level is missing at all. Ultrastructural and cytochemical investigations were undertaken to detect virus particles and the associated cytopathic effects in field-grown grapevine showing different symptom severity. Asymptomatic greenhouse-grown grapevines, which tested negative for GPGV by real time RT-PCR, were sampled as controls. Multiplex real-time RT-PCR and ELISA tests excluded the presence of viruses included in the Italian certification program both in field-grown and greenhouse-grown grapevines. Conversely, evidence was found for ubiquitous presence of Grapevine Rupestris Stem Pitting-associated Virus (GRSPaV), Hop Stunt Viroid (HSVd), and Grapevine Yellow Speckle Viroid 1 (GYSVd-1) in both plant groups. Moreover, in every field-grown grapevine, GPGV was detected by real-time RT-PCR. Ultrastructural observations and immunogold labelling assays showed filamentous flexuous viruses in the bundle sheath cells, often located inside membrane-bound organelles. No cytological differences were observed among field-grown grapevine samples showing different symptom severity. GPGV localization and associated ultrastructural modifications are reported and discussed, in the perspective of assisting management and control of the disease.     

Health Status (Virus) of Native North American Humulus lupulus in the Natural Habitat

Ninety-one native North American Humulus lupulus plants from natural habitats in seven western and mid-western states of the U.S.A. were tested by ELISA serology for presence of two ilarviruses and three carlaviruses common to cultivated hops. All plants in natural habitats were free of detectable viruses. Propagations of 14 such plants from earlier collections had become infected, particularly with two carlaviruses (hop latent virus, American hop latent virus) after exposure for 22 years tobreeding nurseries. ELISA tests of some 284 hop plants primarily from breeding nurseries in Oregon indicated the following infection rates: Prunus necrotic ringspot virus, 85/284, 30%; apple mosaic virus, 88/234, 38%; hop mosaic virus, 59/158, 37 %; hoplatent virus, 104/158, 66 %; and American hop latent virus, 79/158, 50 %. Inoculum reservoirs of AHLV were sought among 53 principally perennial non-Humulus plant species surrounding AHLV-infected hop yards and nurseries. AHLV was neither indigenous to native North American H. lupulus nor detectable in these selected non-Humulus plant species. Breeding nurseries and commercial hop yards, thus, were the only detectable inoculum reservoir for AHLV.     

Development of immunodiagnostics for the detection of <Emphasis Type="Italic">Grapevine leafroll</Emphasis>-<Emphasis Type="Italic">associated virus</Emphasis> 3 (GLRaV-3) in grapevine using in vitro expression and purification of its coat protein gene

A previously cloned coat protein (CP) gene of Grapevine leafroll-associated virus 3 (GLRaV-3) from cultivar Cabernet Souvignon was over-expressed in Escherichia coli strain BL21 expression system as ~ 43 kDa fusion protein containing polyhistidine tag (6His) at its N terminal. The protein was purified from insoluble fraction and reacted positively in western blotting with commercial anti GLRaV-3 polyclonal antiserum (Bioreba, Switzerland) and hence, used as immunogen for the production of polyclonal antisera in New Zealand white rabbits. Polyclonal antiserum specific to GLRaV-3 detected the virus by double antibody sandwich enzyme linked immunosorbent assay using commercial alkaline phosphatase (ALP) conjugated globulin fraction (Bioreba, Switzerland) in GLRaV-3 positive grapevine samples. The immunoreactivity of the antiserum was confirmed through western blotting. The purified antiserum was conjugated with ALP. The primary antiserum along with ALP conjugate successfully detected the GLRaV-3 from the infected sample at 1:8000 and 1:10,000 dilutions, respectively. To the best of our knowledge, it is the first global study wherein the CP of GLRaV-3 was cloned in pET28a(+) expression vector having many advantages over the earlier used expression vectors. The cloned CP gene was expressed, purified and subjected to the production of immunoreagents. The developed immunoreagents will be useful for certification programmes as well as for large scale virus screening to produce GLRaV-3 free grapevines. The indigenously developed immunereagents will provide a cost-effective way of managing grapevine leafroll disease in Indian sub-continent.     

Phage formation in Staphylococcus muscae cultures. X. The relationship between virus synthesis,the release of bacterial ribonucleic acid,virus liberation,and cellular lysis

1. Under a variety of conditions in which cells are infected with one or a few virus particles and the host cells are killed, but no infective particles or virus material is formed as indicated by plaque count, one-step growth curve, or protein or desoxyribonucleic determinations, the cells neither lyse nor release ribonucleic acid into the medium. 2. The "killing" effect of S. muscae phage is separate from its lytic property. 3. The release of ribonucleic acid into the medium is not simply due to the killing of the cell by the virus, and ribonucleic acid is never found in the medium unless virus material is synthesized. 4. Infected cells of S. muscae synthesizing virus release ribonucleic acid into the medium before cellular lysis begins and before any virus is liberated. 5. The higher the phage yield the more ribonucleic acid is released into the medium before any virus is released. 6. Phage may be released from one strain of Staphylococcus muscae without cellular lysis, although bacterial lysis begins shortly after the virus is released. In another strain, infected under similar conditions, virus liberation occurs simultaneously with cellular lysis. 7. The viruses liberated from both bacterial strains appear to be the same in so far as they cannot be distinguished by serological tests, have the same plaque type and plaque size, and need the same amino acids added to the medium in order to grow. Furthermore, the virus liberated from one strain can infect and multiply in the other strain and vice versa. 8. It is suggested that virus synthesis, in S. muscae cells infected with one or a few phage particles, leads to a disturbance of the normal cellular metabolism, resulting in lysis of the host cell.     

Detection of Tomato Ringspot Nepovirus and a Clostero-like Virus in French Hybrid Vidal 256 Grapevines

Eight- to ten-year old French hybrid Vidal 256 grapevines in southern Maryland produced berries about one-third normal size but did not express any obvious leaf symptoms. Electron microscopy of negatively stained tissue-dip preparations and sectioned material from such vines showed individual and membrane-associated 28 nm spherical virus-like particles and closteroviruslike particles. The spherical particles were characterized as an isolate of tomato ringspot virus (TomRSV-G) that infected a wide range of herbaceous hosts by mechanical inoculation, but did not infect tomato, bean or petunia plants susceptible to the type strain of TomRSV. The closterovirus-like particles did not react, by immunosorbent electron microscopy, with antisera to grapevine virus A (grapevine stem-pitting associated virus of Conti et al. 1980) or the 2200 nm Swiss grapevine leafroll closterovirus (Gugleri et al. 1984).     

Membrane transport of antiviral drugs in plants: an electrophysiological study in grapevine explants infected by Grapevine leafroll associated virus 1

Antiviral drugs have demonstrated significant therapeutic potential against phytoviruses, such as inosine monophosphate dehydrogenase inhibitors like mycophenolic acid or thiopurines. However, drug delivery across cellular barriers is a challenging task that calls for investigation. In this study, membrane transport of three antiviral drugs such as mycophenolic acid (MPA), 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) was investigated in order to determine the mechanism involved in transmembrane flux. Electrophysiological tests were carried out on leaves of in vitro grapevine explants naturally infected by Grapevine leafroll associated virus 1 as well as virus-free explants treated with antiviral drugs. Results indicate that membrane electrical responses of the tested antiviral dugs are supported by the metabolism, and virus infection did not cause differences in membrane potential tests. The extent of depolarization was slight for MPA and 6-TG and greater for 6-MP. Complete repolarization always occurred and, in many cases, the electrical potential was greater than the starting level (overshoot). Treatments at 5?°C or in presence of CCCP, an uncoupler of oxidative phosphorylation, led to inhibition of drug effects of the membrane potential, suggesting a metabolic dependence of the transport. However, treatments carried out under sodium orthovanadate, a specific inhibitor of H⁺-ATPase, prove, although only partially, the activity of a proton pump. The complex of electrophysiological tests highlights that drugs such as MPA, 6-MP and 6-TG, when applied to infected or healthy grapevine leaf segments, lead to clear, biphasic, electrical membrane response suggesting an operation of H⁺ co-transport through systems that link substrate translocation across the membrane to the free energy available in a proton electrochemical potential difference.     

The detection of three viruses of hop (Humulus lupulus) by enzyme-linked immunosorbent assay (ELISA)

The recently described technique of enzyme-linked immunosorbent assay (ELISA) was used throughout the 1976 growing season to detect hop mosaic, arabis mosaic and prunus necrotic ringspot viruses in hop plants. On each occasion virus was detected quickly, conveniently and with great sensitivity. The technique was particularly suitable for processing numerous samples collected from the field. Serious difficulties and limitations were encountered in testing comparable material by established techniques. The serology test for the hop strain of arabis mosaic virus by double diffusion in agar gels was very insensitive and only worked satisfactorily early in the growing season. Grafting sensitive Golding hop varieties to detect hop mosaic virus was inconvenient and time-consuming and symptom expression was so slow and erratic that glasshouse space was utilized for long periods. It became impossible to detect prunus necrotic ringspot virus by sap inoculations to cucumber during an exceptionally hot period in mid-summer. The possibilities are discussed of exploiting the ELISA technique for use in large scale surveys and epidemiological studies on viruses of hop and other crops. Changes in the current methods of handling and extracting leaves are considered for increasing the throughput of samples.     

The Complete Sequence of a West Nile Virus Lineage 2 Strain Detected in a Hyalomma marginatum marginatum Tick Collected from a Song Thrush (Turdus philomelos) in Eastern Romania in 2013 Revealed Closest Genetic Relationship to Strain Volgograd 2007

In this study the first complete sequence of the West Nile virus (WNV) lineage 2 strain currently circulating in Romania was determined. The virus was detected in a Hyalomma marginatum marginatum tick collected from a juvenile song thrush (Turdus philomelos) in the Romanian Danube Delta close to the city of Tulcea, end of August 2013. Our finding emphasizes the role of ticks in introduction and maintenance of WNV infections. Sequence analyses revealed close genetic relationship of the Romanian WNV strain to strain Reb_Volgograd_07_H, which was isolated from human brain tissue during an outbreak of West Nile neuroinvasive disease (WNND) in Russia in 2007. In 2010 the Eastern European lineage 2 WNV caused an outbreak of human WNND in Romania. Partial sequences from subsequent years demonstrated that this WNV strain became endemic in Eastern Europe and has been causing outbreaks of varying sizes in southern Russia since 2007 and in Romania since 2010.     

Antiviral activity of Basidiomycete mycelia against influenza type A (serotype H1N1) and herpes simplex virus type 2 in cell culture

In this study, we investigated the in vitro antiviral activity of the mycelia of higher mushrooms against influenza virus type A(serotype H1N1) and herpes simplex virus type 2(HSV-2), strain BH. All 10 investigated mushroom species inhibited the reproduction of influenza virus strain A/FM/1/47(H1N1) in MDCK cells reducing the infectious titer by 2.0–6.0 lg ID50. Four species, Pleurotus ostreatus, Fomes fomentarius, Auriporia aurea, and Trametes versicolor, were also determined to be effective against HSV-2 strain BH in RK-13 cells, with similar levels of inhibition as for influenza. For some of the investigated mushroom species—Pleurotus eryngii, Lyophyllum shimeji, and Flammulina velutipes—this is the first report of an anti-influenza effect. This study also reports the first data on the medicinal properties of A. aurea, including anti-influenza and antiherpetic activities. T. versicolor 353 mycelium was found to have a high therapeutic index(324.67), and may be a promising material for the pharmaceutical industry as an anti-influenza and antiherpetic agent with low toxicity. Mycelia with antiviral activity were obtained in our investigation by bioconversion of agricultural wastes(amaranth flour after CO2 extraction), which would reduce the cost of the final product and solve some ecological problems.     

Transient expression of artificial microRNAs targeting Grapevine fanleaf virus and evidence for RNA silencing in grapevine somatic embryos

Grapevines are affected worldwide by viruses that compromise fruit yield and quality. Grapevine fanleaf virus (GFLV) causes fanleaf degeneration disease, a major threat to grapevine production. Transgenic approaches exploiting the RNA silencing machinery have proven suitable for engineering viral resistance in several crop species. However, the artificial microRNA (amiRNA)-based strategy has not yet been reported in grapevine. We developed two amiRNA precursors (pre-amiRNAs) targeting the coat protein (CP) gene of GFLV and characterised their functionality in grapevine somatic embryos. To create these pre-amiRNAs, natural pre-miR319a of Arabidopsis thaliana was modified by overlapping PCR in order to replace miR319a with two amiRNAs targeting different regions of the CP gene: amiR^CP-1 or amiR^CP-2. Transient expression of these two pre-amiRNA constructs was tested in grapevine somatic embryos after co-cultivation with Agrobacterium tumefaciens. Expression of amiR^CP-1 and amiR^CP-2 was detected in plant tissues by an endpoint stem-loop RT-PCR as early as 1?day after a 48-h co-cultivation, indicating active processing of pre-amiRNAs by the plant machinery. In parallel, GUS-sensor constructs (G^CP-1 and G^CP-2) were obtained by fusing the target sequence of amiR^CP-1 or amiR^CP-2 to the 3?? terminus of the GUS gene. Co-transformation assays with GUS-sensors and the pre-amiRNA constructs provided evidence for in vivo recognition and cleavage of the 21-nt target sequence of GUS-sensors by the corresponding amiRNA. This is the first report of amiRNA ectopic expression in grapevine. The constructs we developed could be useful for engineering GFLV-resistant grapes in the future.     

Hop line-pattern virus in relation to the etiology and distribution of nettlehead disease

Hop line-pattern virus (HLPV) was transmissible by mechanical inoculation to hop plants; it induced characteristic severe symptoms in Humulus lupulus L. var. neo-mexicanus Nels. & Cockerell and the commercial derivatives College Cluster and Keyworth's Midseason, but none in the traditional English varieties of H. lupulus (e.g. Fuggle).
Mechanical transmission of hop nettlehead virus (HNV) was facilitated by the presence of HLPV in the test plants; hop seedlings and clonal plants escaped infection by sap inoculum that infected plants of two varieties already infected with HLPV. HNV was also transferred by stem contact and by knife cuts to plants carrying HLPV.
Infection with HLPV was latent in twelve nettlehead-diseased Fuggle plants from different fields, and in diseased and symptomless plants in a nettlehead outbreak in W.G.V., a variety that previously had escaped infection. It is suggested either that HLPV predisposes hop plants to infection with HNV or that nettlehead disease is caused by dual infection with both viruses.
Localized and scattered patterns of nettlehead spread were observed in hop plantations; these two types are usually attributed to different modes of spread which would be compatible with a complex etiology of the disease.